ASYMMETRIC siRNA FOR INHIBITING EXPRESSION OF MALE PATTERN HAIR LOSS TARGET GENE

ABSTRACT

The present invention relates to an asymmetric siRNA which inhibits an expression of male pattern hair loss target genes and a use thereof and, more particularly, to an asymmetric siRNA which inhibits an expression of 3-oxo-5-alpha-steroid-4-dehydrogenase 1 (SRD5A1) gene, 3-oxo-5-alpha-steroid-4-dehydrogenase 2 (SRD5A2) gene or androgen receptor (AR) gene, and a composition for prevention or treatment of hair loss comprising the asymmetric siRNA.

TECHNICAL FIELD

The present invention relates to asymmetric siRNA for inhibiting theexpression of a target gene for male pattern hair loss and a usethereof, and more particularly to asymmetric siRNA for inhibiting theexpression of the 3-oxo-5-alpha-steroid 4-dehydrogenase 1 (SRD5A1) gene,the 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2) gene, or theandrogen receptor (AR) gene, and a composition for preventing ortreating hair loss which comprises the asymmetric siRNA.

BACKGROUND ART

Human hair is formed in hair follicles. There are papillae in the hairfollicles, small blood vessels are distributed in the papillae to supplynutrients necessary for hair growth, and sebaceous glands aredistributed on the upper ends of side surfaces of the papillae tosecrete sebum to protect the hair. The dermal papilla regulates hairgrowth and is the site where male hormones act in male-pattern hairloss. The hair matrix is a site where cell division occurs under thecontrol of the dermal papilla and hair grows.

The main factor of male hair loss is due to the effect of abnormalhormones. At puberty, sex hormones are actively secreted and thesecondary sexual character appears. These changes are caused by sexhormones, i.e., androgens (male hormones) and estrogens (femalehormones). Androgens develop body hair under the eyebrows, and estrogenmainly promotes hair growth. For men, hair loss is due to excessivesecretion of androgens which results in inhibition of the action ofestrogen by the excessively secreted androgens.

Specifically, steroid 5-alpha reductase is involved in the male hairloss mechanism by male hormones, and steroid 5-alpha reductase is a mainenzyme that reduces testosterone, which is a male hormone, to DHT(dihydrotestosterone). The resulting DHT is known to bind to an androgenreceptor to thereby regulate hair growth in the hair follicles and beinvolved in the proliferation of sebaceous glands.

The androgen receptor is a male hormone (androgen) receptor and is knownto be capable of binding to both testosterone and DHT, but have astronger binding affinity with DHT. It is known that the inhibition ofsteroid 5-alpha reductase and an androgen receptor increases hair growthfactors and induces hair growth, whereas the activation of steroid5-alpha reductase and an androgen receptor inhibits hair growth,resulting in the occurrence of hair loss (Chhipa, R R et al., Prostate,73:1483, 2013; Azzouni, F et al, Advances in Urology, 2012:18, 2012;Winiarska, A. et al., Skin Pharmacology and Physiology, 19:311, 2006).

There are two types of steroid 5-alpha reductase: type 1 and type 2.Steroid 5-alpha reductase type 1 is mainly distributed throughout theskin, especially in the sebaceous glands, and steroid 5-alpha reductasetype 2 is mainly distributed around the dermal papilla of the hairfollicles and in the outer root sheath. In the early stage of drugdevelopment, hair loss therapeutic agents targeting only steroid 5-alphareductase type 2 have mainly been developed, but therapeutic agents forinhibiting both steroid 5-alpha reductase type 1 and type 2 haverecently been developed since the type 1 also has been found to affecthair growth.

Among these, finasteride may be used as a drug for inhibiting steroid5-alpha reductase type 2. Finasteride was originally developed as atherapeutic agent for benign prostatic hypertrophy, it has been approvedby the FDA and the Korean Food and Drug Administration as a male patternhair loss therapeutic agent since finasteride was confirmed to promotehair growth in patients administered. Dutasteride is known to be atherapeutic ingredient that inhibits both steroid 5-alpha reductase type1 and type 2. Drugs which bind to the androgen receptor and thus acts asan antagonist that hinders the binding between the androgen receptor andDHT are called anti-androgen drugs, and as these anti-androgen drugs,Cimetidine, Spironolactone, Flutamide, Cyproterone acetate, and the likeare known.

However, these therapeutic ingredients have problems such as sexualdysfunction, fatigue appeal, and the like, and the use thereof islimited in women of childbearing age. These may cause fetalmalformations when exposed to pregnant women. Therefore, there is a needto develop a therapeutic agent for hair loss without such side effects.

Under these technical backgrounds, the inventors of the presentinvention confirmed that a novel RNAi drug with minimal side effectsdeveloped using siRNA for inhibiting the expression of the3-oxo-5-alpha-steroid 4-dehydrogenase 1 (SRD5A1) gene, the3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2) gene, or the androgenreceptor (AR) gene was able to exhibit a desired effect of preventing ortreating hair loss, and thus completed the present invention.

DISCLOSURE Technical Problem

It is an object of the present invention to provide asymmetric shorterduplex siRNA (asiRNA) specifically binding to a SRD5A1-encoding gene, aSRD5A2-encoding gene, or an AR-encoding gene.

It is another object of the present invention to provide a compositionfor preventing or treating hair loss which comprises the asiRNA, or amethod of preventing or treating hair loss.

Technical Solution

To achieve the above object, the present invention provides siRNAspecifically binding to mRNA of a 3-oxo-5-alpha-steroid 4-dehydrogenase1 (SRD5A1)-encoding gene, mRNA of a 3-oxo-5-alpha-steroid4-dehydrogenase 2 (SRD5A2)-encoding gene, or mRNA of an androgenreceptor (AR)-encoding gene and comprising a sense strand having alength of 15-17 nt and an antisense strand complementary to the sensestrand and having a length of 19 nt or more, wherein the 3′-terminus ofthe sense strand and the 5′-terminus of the antisense strand form ablunt end.

The present invention also provides a composition for preventing ortreating hair loss which comprises the siRNA.

The present invention also provides a method of preventing or treatinghair loss, comprising administering the siRNA to a subject.

The present invention also provides a use of the siRNA for preventing ortreating hair loss.

The present invention also provides a use of the siRNA for preparing adrug for the prevention or treatment of hair loss.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B illustrate results showing the gene inhibitoryefficiency of asiRNA against 100 sequences targeting SRD5A1. HuH-7 cellswere transfected with 0.3 nM asiRNA targeting each nucleotide sequence,and after 24 hours, the expression level of SRD5A1 mRNA was measuredusing qRT-PCR, and the graphs show the mean and SD of two repeatedexperiments.

FIG. 2 illustrates results showing the gene inhibitory efficiency ofasiRNA against 12 sequences targeting SRD5A1. HuH-7 cells weretransfected with 0.3 nM, 1 nM, or 10 nM asiRNA targeting each nucleotidesequence, and after 48 hours, the expression level of the SRD5A1 proteinwas measured by western blotting.

FIG. 3 illustrates results showing the gene inhibitory efficiency of 12kinds of cp-asiRNAs targeting SRD5A1 and having various chemicalmodifications added thereto, through two repeated experiments. HuH-7cells were incubated with 1 μM or 3 μM cp-asiRNA targeting eachnucleotide sequence, and after 48 hours, the expression level of theSRD5A1 protein was measured by western blotting.

FIG. 4 illustrates results showing the gene inhibitory efficiency of 2kinds of cp-asiRNA targeting SRD5A1 and having various chemicalmodifications added thereto. HuH-7 cells were incubated with 0.3 μM, 1μM, or 3 μM cp-asiRNA targeting each nucleotide sequence, and after 24hours, the expression level of SRD5A1 mRNA was measured using real-timePCR, and the graph shows the mean and SD of four repeated experiments.

FIGS. 5A and 5B illustrate results showing the gene inhibitoryefficiency of asiRNA against 112 sequences targeting SRD5A2. HuH-7 cellswere transfected with 0.3 nM asiRNA targeting each nucleotide sequence,and after 24 hours, the expression level of SRD5A2 mRNA was measuredusing qRT-PCR, and the graphs show the mean and SD of two repeatedexperiments.

FIG. 6A illustrates results showing the gene inhibitory efficiency ofasiRNA against 23 sequences targeting SRD5A2, and FIG. 6B illustratesresults showing the gene inhibitory efficiency of asiRNA against 6sequences and 4 sequences, which target SRD5A2. HuH-7 cells weretransfected with 3 nM or 10 nM of asiRNA targeting each nucleotidesequence, and after 48 hours, the expression level of the SRD5A2 proteinwas measured by western blotting.

FIG. 7 illustrates results showing the gene inhibitory efficiency of 12kinds of cp-asiRNA targeting SRD5A2 and having various chemicalmodifications added thereto, through two repeated experiments. HuH-7cells were incubated with 1 μM of cp-asiRNA targeting each nucleotidesequence, and after 48 hours, the expression level of the SRD5A2 proteinwas measured by western blotting.

FIG. 8 illustrates results showing the gene inhibitory efficiency of 2selected kinds of cp-asisRD5A2. HuH-7 cells were incubated withcp-asiRNA targeting each nucleotide sequence at a concentration rangingfrom 1.95 nM to 1,000 nM, and after 24 hours, the expression level ofSRD5A2 mRNA was measured by RT-PCR.

FIG. 9 illustrates results showing the gene inhibitory efficiency of 2selected kinds of cp-asiSRD5A2. HuH-7 cells were incubated with 0.1 μM,0.3 μM, 1 μM, or 3 μM of cp-asiRNA targeting each nucleotide sequence,and after 48 hours, the expression level of the SRD5A2 protein wasmeasured by western blotting.

FIGS. 10A and 10B illustrate results showing the gene inhibitoryefficiency of asiRNA against 118 sequences targeting AR. A549 cells weretransfected with 0.3 nM of asiRNA targeting each nucleotide sequence,and after 24 hours, the expression level of AR mRNA was measured throughqRT-PCR, the graphs showing the mean and SD of two repeated experiments.

FIG. 11 illustrates results showing the inhibitory efficiency of asiRNAagainst 20 sequences targeting AR at a protein level. A549 cells weretransfected with 0.3 nM asiRNA targeting each nucleotide sequence, andafter 48 hours, the expression level of the AR protein was measured bywestern blotting, and experiments were repeated three times.

FIG. 12 illustrates results showing the gene inhibitory efficiency ofasiRNA against 9 sequences targeting AR. A549 cells were transfectedwith 0.1 nM asiRNA targeting each nucleotide sequence, and after 48hours, the expression levels of AR mRNA and the AR protein were measuredby qRT-PCR and western blotting, respectively.

FIG. 13 illustrates results showing the gene inhibitory efficiency of 9kinds of cp-asiRNA targeting an AR and having various chemicalmodifications added thereto. A549 cells were incubated with 1 μM or 3 μMof cp-asiRNA targeting each nucleotide sequence, and after 48 hours, theexpression level of AR mRNA was measured through real-time PCR, and thegraph shows the mean and SD of four repeated experiments.

FIG. 14 illustrates results showing the gene inhibitory efficiency of 9kinds of cp-asiRNA targeting an AR and having various chemicalmodifications added thereto. A549 cells were incubated with 1 μM ofcp-asiRNA targeting each nucleotide sequence, and after 48 hours, theexpression level of the AR protein was measured using a western blottingassay.

DETAILED DESCRIPTION AND EXEMPLARY EMBODIMENTS

Unless otherwise defined, all technical and scientific terms as usedherein have the same meanings as those commonly understood by one ofordinary skill in the art to which the present invention pertains.Generally, the nomenclature used herein is well known and commonly usedin the art.

Accordingly, in one aspect, the present invention relates to siRNAspecifically binding to mRNA of a 3-oxo-5-alpha-steroid 4-dehydrogenase1 (SRD5A1)-encoding gene having SEQ ID NO: 678, mRNA of a3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2)-encoding gene havingSEQ ID NO: 679, or mRNA of an androgen receptor (AR)-encoding genehaving SEQ ID NO: 680 and comprising a sense strand having a length of15-17 nt and an antisense strand complementary to the sense strand andhaving a length of 19 nt or more, wherein the 3′-terminus of the sensestrand and the 5′-terminus of the antisense strand form a blunt end.

The SRD5A1-encoding gene, which is a target gene for male pattern hairloss, for example, androgenetic alopecia, has mRNA Accession Number:NM_001047.3, NM_001324322.1, or NM_001324323.1, which respectivelyinclude sequences with SEQ ID: 669, SEQ ID NO: 670, and SEQ ID NO: 671.The SRD5A2-encoding gene has mRNA Accession Number: NM_000348.3 andincludes a sequence having SEQ ID NO: 672. The AR-encoding gene has mRNAAccession Number: NM_001011645.2 and includes a sequence having SEQ IDNO: 673.

In the present invention, siRNA is a concept including all substanceshaving a general RNA interference (RNAi) action. RNAi is anintracellular mechanism for gene regulation that was first found inCaenorhabditis elegans in 1998, and as for the mechanism action, it isknown that the antisense strand of a double-stranded RNA introduced intoa cell complementarily binds to mRNA of a target gene to thereby inducethe degradation of the target gene. In this regard, small interferingRNA (siRNA) is one of the methods of inhibiting gene expression invitro. siRNAs of 19-21 bp in length are theoretically capable ofperforming selective inhibition against almost all genes, and thus canbe developed as therapeutic agents for various gene-related diseasessuch as cancer, viral infection, and the like, and is the new candidatedrug development technology that has recently drawn the most attention.The first attempt to perform in vivo treatment using siRNA in mammalswas in mid-2003, and since then, there have been numerous reports of invivo treatment thanks to many attempts for application studies.

However, contrary to the possibility of in vivo treatment, side effectsand disadvantages of siRNA have continually been reported. To develop anRNAi-based therapeutic agent, challenges such as: 1) the absence of aneffective delivery system; 2) the off-target effect; 3) the induction ofimmune responses; and 4) intracellular RNAi mechanism saturation need tobe overcome. Although siRNAs are an effective method of directlyregulating target gene expression, it is difficult to develop atherapeutic agent using such siRNAs due to the above-described problems.With regard thereto, the applicant of the present invention hasdeveloped an asymmetric shorter duplex siRNA (asiRNA) structure-relatedtechnology (WO2009/078685). asiRNA is an asymmetric RNAi-inducingstructure having a shorter double helix length than the 19+2 structureof existing siRNAs. asiRNA is a technology that has overcome knownproblems with the existing siRNA structure technology, such as theoff-target effect, RNAi mechanism saturation, immune responses by TLR3,and the like, and accordingly is used for the development of a new RNAidrug with minimal side effects.

Based on this, the present invention provides asymmetric siRNA includinga sense strand having a length of 15-17 nt and an antisense strandcomplementary to the sense strand and having a length of 19 nt or more,and thus the siRNA according to the present invention may stablymaintain high delivery efficiency without incurring problems such as theoff-target effect, RNAi mechanism saturation, immune responses by TLR3,and the like, and may inhibit the expression of a 5α-reductase type 1target gene, a 5α-reductase type 2 target gene, and an androgen receptortarget gene.

In the present invention, the term “sense strand” refers to apolynucleotide having the same nucleic acid sequence as that of theSRD5A1-, SRD5A2-, or AR-encoding gene, and has a length of 15-17 nt. Inone embodiment, the sense strand may have a length of 15 nt, 16 nt, or17 nt.

The inventors of the present application selected, as target genes,5α-reductase type 1, 5α-reductase type 2, and an androgen receptor,which play a major role in inhibiting the synthesis of proteins requiredfor hair follicle growth in male pattern hair loss and inducing hairloss by reducing the dermal papilla. As a result of screening 100 ormore siRNAs targeting each target gene and selecting siRNAs withexcellent inhibitory efficiency from among the same, it was confirmedthat siRNA comprising a sense strand having one selected from SEQ IDNOS: 5, 6, 15, 18, 40, 48, 49, 59, 62, 69, 77, 86, 205, 208, 228, 231,232, 233, 237, 238, 239, 240, 242, 248, 249, 259, 260, 262, 265, 283,284, 285, 291, 292, 300, 471, 477, 498, 500, 502, 503, 505, 506, 507,509, 510, 515, 517, 518, 521, 524, 534, 538, 539, and 546 and anantisense strand complementary to the sense strand, effectively reducedthe expression of mRNA of the SRD5A1-, SRD5A2-, or AR-encoding gene.

Specifically, siRNA comprising a sense strand having SEQ ID NO: 5, 6,15, 18, 40, 48, 49, 59, 62, 69, 77, or 86 and an antisense strandcomplementary to the sense strand, may reduce the expression of mRNA ofthe SRD5A1-encoding gene, siRNA comprising a sense strand having SEQ IDNO: 205, 208, 228, 231, 232, 233, 237, 238, 239, 240, 242, 248, 249,259, 260, 262, 265, 283, 284, 285, 291, 292, or 300 and an antisensestrand complementary to the sense strand, may reduce the expression ofmRNA of the SRD5A2-encoding gene, and siRNA comprising a sense strandhaving SEQ ID NO: 471, 477, 498, 500, 502, 503, 505, 506, 507, 509, 510,515, 517, 518, 521, 524, 534, 538, 539, or 546 and an antisense strandcomplementary to the sense strand, may reduce the expression of mRNA ofthe AR-encoding gene.

Specifically, it was confirmed that siRNA comprising a sense strandhaving one selected from the group consisting of SEQ ID NOS: 48, 49, 69,86, 231, 259, 260, 262, 498, 500, 506, 509, 510, 518, 538, 539, and 546and an antisense strand complementary to the sense strand alsoeffectively inhibited the expression of the SRD5A1 protein, the SRD5A2protein, or the AR protein.

The 3′-terminus of the sense strand and the 5′-terminus of the antisensestrand form a blunt end. For example, the 5′-terminus of the antisensestrand may include, for example, an overhang of 1 nt, 2 nt, 3 nt, 4 nt,5 nt, 6 nt, 7 nt, or 8 nt.

In the present invention, the antisense strand is a polynucleotidecomplementary to the target gene and has a length of 19 nt or more, forexample, 20 nt or more, 21 nt or more, 22 nt or more, 23 nt or more, 24nt or more, 25 nt or more, 26 nt or more, 27 nt or more, 29 nt or more,30 nt or more, or 31 nt or more. In one embodiment, the antisense strandmay have a length between 19 nt and 24 nt, for example, 19 nt, 20 nt, 21nt, 22 nt, 23 nt, or 24 nt. The antisense strand may have a sequencepartially complementary to the sense strand in consideration of theasymmetric structure.

The antisense strand may, for example, be selected from the groupconsisting of SEQ ID NOS: 105, 106, 115, 118, 140, 148, 149, 159, 162,169, 177, 186, 317, 320, 340, 343, 344, 345, 349, 350, 351, 352, 354,360, 361, 371, 372, 374, 377, 395, 396, 397, 403, 404, 412, 589, 595,616, 618, 620, 621, 623, 624, 625, 627, 628, 633, 635, 636, 639, 642,652, 656, 657, and 664.

Specifically, it was confirmed that siRNA comprising an antisense strandselected from the group consisting of SEQ ID NOS: 148, 149, 169, 186,343, 371, 372, 374, 616, 618, 624, 627, 628, 636, 656, 657, and 664 alsoeffectively inhibited the expression of the SRD5A1 protein, the SRD5A2protein, or the AR protein.

In some embodiments, the sense strand or antisense strand of the siRNAmay include one or more chemical modifications.

General siRNAs are unable to penetrate through the cell membrane due toreasons such as high negative charge, high molecular weight, and thelike, and are rapidly degraded and eliminated in the blood, making itdifficult to deliver an amount sufficient for RNAi induction to anactual target site. Currently, in the case of in vitro delivery,numerous high-efficiency delivery methods using cationic lipids andcationic polymers have been developed, but in vivo delivery of siRNA asefficient as in vitro delivery thereof is difficult, and siRNA deliveryefficiency is reduced by interactions between various proteins presentin the living body.

Therefore, the inventors of the present application developed cellpenetrating asiRNA (cp-asiRNA) having self-transfer ability that enableseffective intracellular delivery without a separate delivery vehicle byintroducing a chemical modification into an asymmetric siRNA structure.

The chemical modification in the sense strand or the antisense strandmay comprise, for example, at least one selected from the groupconsisting of:

a modification in which an —OH group at the 2′ carbon position of asugar structure in a nucleotide is substituted with —CH₃ (methyl), —OCH₃(methoxy), —NH₂, —F (fluorine), —O-2-methoxyethyl-O-propyl,—O-2-methylthioethyl, —O-3-aminopropyl, or —O-3-dimethylaminopropyl;

a modification in which oxygen in a sugar structure in a nucleotide issubstituted with sulfur;

a modification of a nucleotide bond to a phosphorothioate,boranophosphate or methyl phosphonate;

a modification to peptide nucleic acid (PNA), locked nucleic acid (LNA),or unlocked nucleic acid (UNA); and

cholesterol or cell-penetrating peptide binding.

In one embodiment, the chemical modification in the sense or antisensestrand may be substitution of an —OH group at the 2′ carbon position ofa sugar structure in a nucleotide with —CH₃ (methyl), modification of anucleotide bond into phosphorothioate, or cholesterol binding. This mayenhance the in vivo stability of siRNA.

When the —OH group at the 2′ carbon position of a sugar structure issubstituted with —CH₃ (methyl) or when the nucleotide bond is modifiedinto a phosphorothioate, resistance to nucleases may be increased, andbinding to the cell membrane via cholesterol binding may facilitate theintracellular delivery of siRNA.

In particular, the chemical modification may include at least onemodification selected from the group consisting of: a modification inwhich an —OH group at the 2′ carbon position of a sugar structure in the5′- or 3′-terminus nucleotide of the sense strand is substituted with—CH₃ (methyl); a modification in which an —OH group at the 2′ carbonposition of a sugar structure in two or more nucleotides of the sensestrand or the antisense strand is substituted with —CH₃ (methyl); amodification of 25% or more of nucleotides bonds in the sense orantisense strand to phosphorothioate; and cholesterol binding at the3′-terminus of the sense strand.

With regard to the modification in which an —OH group at the 2′ carbonposition of a sugar structure in a nucleotide is substituted with —CH₃(methyl), the —OH group at the 2′ carbon position of the sugar structurein a nucleotide positioned at the 5′-terminus of the sense strand may besubstituted with —CH₃ (methyl). In addition, a 2′-O-methylatednucleoside, in which an —OH group at the 2′ carbon position of a sugarstructure is substituted with —CH₃ (methyl), may be continuously ordiscontinuously included in a 5′-terminus to 3′-terminus direction ofthe sense strand. 2′-O-methylated nucleosides and unmodified nucleosidesmay be alternately included in the sense strand. 2, 3, 4, 5, 6, 7, or 8consecutive 2′-O-methylated nucleosides and unmodified nucleosides maybe alternately included in the sense strand. For example, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, 2 to 8, or 8 2′-O-methylated nucleosides may be presentin the sense strand.

2′-O-methylated nucleosides may be continuously or discontinuouslyincluded in a 5′-terminus to 3′-terminus of the antisense strand.2′-O-methylated nucleosides and unmodified nucleotides may bealternately included in the antisense strand. 2, 3, 4, 5, 6, 7, or 8consecutive 2′-O-methylated nucleosides and unmodified nucleosides maybe alternately included in the antisense strand. For example, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, or 2-7 2′-O-methylated nucleosides may be presentin the antisense strand.

With regard to the modification of a nucleotide bond to aphosphorothioate, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% of bonds between ribonucleotides in thesense strand may be modified into phosphorothioate. In some embodiments,all (100%) of the bonds between ribonucleotides in the sense strand maybe modified into phosphorothioate.

At least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the bonds between ribonucleotides in the antisensestrand may be modified into phosphorothioate. In some embodiments, atotal (100%) of the bonds between ribonucleotides in the antisensestrand may be modified into phosphorothioate.

In another aspect, the present invention relates to a composition forthe prevention or treatment of hair loss, which comprises the siRNA.

The term “treatment” as used herein means reducing the symptoms of hairloss or the severity of hair loss in a subject to which the compositionis administered or preventing the same from being aggravated, and insome cases may include the progression of hair growth. The term“prevention” as used herein means preventing or delaying the initiationof hair loss, or reducing the possibility of developing hair loss.

The composition may further be prepared including one or morepharmaceutically acceptable carriers, in addition to the siRNA as anactive ingredient. The pharmaceutically acceptable carrier has to becompatible with the active ingredient of the present invention, and maybe one selected from physiological saline, sterile water, Ringer'ssolution, buffered saline, dextrose solution, maltodextrin solution,glycerol, ethanol, and a mixture of two or more of these components. Ifnecessary, the composition may include other general additives such asan antioxidant, a buffer, a bacteriostatic agent, and the like. Inaddition, the composition may be formulated into an injectablepreparation such as an aqueous solution, a suspension, an emulsion, orthe like by further adding a diluent, a dispersing agent, a surfactant,a binder, and a lubricant. In particular, the composition may beformulated into a lyophilized preparation. The lyophilized preparationmay be formulated using a method commonly used in the art to which thepresent invention pertains, and a stabilizer for lyophilization may alsobe added.

An administration method of the composition may be determined by one ofordinary skill in the art on the basis of general symptoms of patientsand the severity of diseases. In addition, the composition may beformulated into various forms such as powders, tablets, capsules,liquids, injections, ointments, syrups, and the like, and may also beprovided in a unit dosage or multiple dosage container, for example,sealed ampoules and vials, and the like.

The composition may be administered orally or parenterally. Theadministration route of the composition according to the presentinvention may be, but is not limited to, for example, oraladministration, intravenous administration, intramuscularadministration, intraarterial administration, intramedullaryadministration, intradural administration, intracardiac administration,transdermal administration, subcutaneous administration, intraperitonealadministration, intestinal administration, sublingual administration, ortopical administration. The dosage of the composition according to thepresent invention varies depending on the body weight, age, gender, andhealth condition of a patient, diet, administration time, administrationmethod, excretion rate, severity of disease, or the like, and may beeasily determined by those of ordinary skill in the art. In addition,for clinical administration, the composition of the present inventionmay be formulated into a suitable form using known techniques.

In another aspect of the present invention, there is provided a methodof preventing or treating hair loss, comprising administering the siRNAto a subject.

In another aspect, the present invention relates to a use of the siRNAfor preventing or treating hair loss.

In another aspect, the present invention relates to a use of the siRNAfor preparing a drug for the prevention or treatment of hair loss.

Configurations included in the prevention or treatment method accordingto the present invention are the same as those included in theaforementioned embodiments, and thus the foregoing description may beequally applied to the prevention or treatment method.

Hereinafter, the present invention will be described in further detailwith reference to the following examples. It will be obvious to those ofordinary skill in the art that these examples are provided forillustrative purposes only and are not intended to limit the scope ofthe present invention.

[Example 1] Screening for 100 Kinds of RNAi-Inducing Double-StrandedNucleic Acid Molecules Targeting SRD5A1

To obtain high-efficiency RNAi-inducing double-stranded nucleic acidmolecules targeting SRD5A1, the target sequence of the SRD5A1 gene wasselected and then asiRNA was designed. The asiRNA structure is differentfrom that of generally known siRNAs, and thus when the nucleotidesequences of asiRNA are designed using a general siRNA design program,it may be somewhat difficult to design an optimized asiRNA. Therefore,asiRNA was constructed by the following method. An NCBI db search wasused to obtain information on the SRD5A1 gene (mRNA Accession Number:NM_001047.3, NM_001324322.1, NM_001324323.1), which is the target genepertaining to male pattern hair loss (androgenetic hair loss). Forsubsequent animal experiments, nucleotide sequences were secured inconsideration of the nucleotide sequence homology with mice, and then100 kinds of asiRNA were designed according to a design method such asthe exclusion of sequences having a GC content of 30-62% and 4 or more Gor C consecutive bases, and then synthesized by OliX Inc. (Korea). Thesynthesized sense and antisense strand RNA oligonucleotides wereannealed at 95° C. for 2 minutes through incubation at 37° C. for 1hour, and the asiRNA annealed by 10% polyacrylamide gel electrophoresis(PAGE) was confirmed using a UV transilluminator.

TABLE 1 100 Kinds of asiRNA nucleotide sequences targeting3-oxo-5-alpha-steroid 4-dehydrogenase 1 100 kinds Sequence (5′-3′) No.Name S AS 1 asiSRD5A1 1 GCAGAUACUUGAGCCA UGGCUCAAGUAUCUG CUUUGC(SEQ ID NO: 1) (SEQ ID NO: 101) 2 asiSRD5A1 2 AAGCAGAUACUUGAGCGCUCAAGUAUCUGCU UUGCAA (SEQ ID NO: 2) (SEQ ID NO: 102) 3 asiSRD5A1 3CAAAGCAGAUACUUGA UCAAGUAUCUGCUUU GCAAAU (SEQ ID NO: 3) (SEQ ID NO: 103)4 asiSRD5A1 4 UGCAAAGCAGAUACUU AAGUAUCUGCUUUGC AAAUAG (SEQ ID NO: 4)(SEQ ID NO: 104) 5 asiSRD5A1 5 GUGCAGUGUAUGCUGA UCAGCAUACACUGCA CAAUGG(SEQ ID NO: 5) (SEQ ID NO: 105) 6 asiSRD5A1 6 UUGUGCAGUGUAUGCUAGCAUACACUGCACA AUGGCU (SEQ ID NO: 6) (SEQ ID NO: 106) 7 asiSRD5A1 7CAUUGUGCAGUGUAUG CAUACACUGCACAAU GGCUCA (SEQ ID NO: 7) (SEQ ID NO: 107)8 asiSRD5A1 8 UUUUGGCUUGUGGUUA UAACCACAAGCCAAA ACCUAU (SEQ ID NO: 8)(SEQ ID NO: 108) 9 asiSRD5A1 9 CGGGCAUGUUGAUAAA UUUAUCAACAUGCCC GUUAAC(SEQ ID NO: 9) (SEQ ID NO: 109) 10 asiSRD5A1 10 AUAUCCUAAGGAAUCUAGAUUCCUUAGGAUA UGAUCU (SEQ ID NO: 10) (SEQ ID NO: 110) 11 asiSRD5A1 11AUCUCAGAAAACCAGG CCUGGUUUUCUGAGA UUCCUU (SEQ ID NO: 11) (SEQ ID NO: 111)12 asiSRD5A1 12 GAAUCUCAGAAAACCA UGGUUUUCUGAGAUU CCUUAG (SEQ ID NO: 12)(SEQ ID NO: 112) 13 asiSRD5A1 13 AGGAAUCUCAGAAAAC GUUUUCUGAGAUUCC UUAGGA(SEQ ID NO: 13) (SEQ ID NO: 113) 14 asiSRD5A1 14 CUGGAUACAAAAUACCGGUAUUUUGUAUCCA GUAUCU (SEQ ID NO: 14) (SEQ ID NO: 114) 15 asiSRD5A1 15UACUGGAUACAAAAUA UAUUUUGUAUCCAGU AUCUCC (SEQ ID NO: 15) (SEQ ID NO: 115)16 asiSRD5A1 16 GAUACUGGAUACAAAA UUUUGUAUCCAGUAU CUCCUG (SEQ ID NO: 16)(SEQ ID NO: 116) 17 asiSRD5A1 17 GAGAUACUGGAUACAA UUGUAUCCAGUAUCU CCUGGU(SEQ ID NO: 17) (SEQ ID NO: 117) 18 asiSRD5A1 18 AGGAGAUACUGGAUACGUAUCCAGUAUCUCC UGGUUU (SEQ ID NO: 18) (SEQ ID NO: 118) 19 asiSRD5A1 19CCAGGAGAUACUGGAU AUCCAGUAUCUCCUG GUUUUC (SEQ ID NO: 19) (SEQ ID NO: 119)20 asiSRD5A1 20 AAUACCAAGGGGAGGC GCCUCCCCUUGGUAU UUUGUA (SEQ ID NO: 20)(SEQ ID NO: 120) 21 asiSRD5A1 21 AAAAUACCAAGGGGAG CUCCCCUUGGUAUUU UGUAUC(SEQ ID NO: 21) (SEQ ID NO: 121) 22 asiSRD5A1 22 GAGGCUUAUUUGAAUAUAUUCAAAUAAGCCU CCCCUU (SEQ ID NO: 22) (SEQ ID NO: 122) 23 asiSRD5A1 23CAGCCAACUAUUUUGG CCAAAAUAGUUGGCU GCAGUU (SEQ ID NO: 23) (SEQ ID NO: 123)24 asiSRD5A1 24 UGCAGCCAACUAUUUU AAAAUAGUUGGCUGC AGUUAC (SEQ ID NO: 24)(SEQ ID NO: 124) 25 asiSRD5A1 25 ACUGCAGCCAACUAUU AAUAGUUGGCUGCAG UUACGU(SEQ ID NO: 25) (SEQ ID NO: 125) 26 asiSRD5A1 26 UAACUGCAGCCAACUAUAGUUGGCUGCAGUU ACGUAU (SEQ ID NO: 26) (SEQ ID NO: 126) 27 asiSRD5A1 27CGUAACUGCAGCCAAC GUUGGCUGCAGUUAC GUAUUC (SEQ ID NO: 27) (SEQ ID NO: 127)28 asiSRD5A1 28 AUGGAGUGGUGUGGCU AGCCACACCACUCCA UGAUUU (SEQ ID NO: 28)(SEQ ID NO: 128) 29 asiSRD5A1 29 UCAUGGAGUGGUGUGG CCACACCACUCCAUG AUUUCU(SEQ ID NO: 29) (SEQ ID NO: 129) 30 asiSRD5A1 30 AAUCAUGGAGUGGUGUACACCACUCCAUGAU UUCUCC (SEQ ID NO: 30) (SEQ ID NO: 130) 31 asiSRD5A1 31GAAAUCAUGGAGUGGU ACCACUCCAUGAUUU CUCCAA (SEQ ID NO: 31) (SEQ ID NO: 131)32 asiSRD5A1 32 GAGAAAUCAUGGAGUG CACUCCAUGAUUUCU CCAAAA (SEQ ID NO: 32)(SEQ ID NO: 132) 33 asiSRD5A1 33 CCCUGGCCAGCUGGUC GACCAGCUGGCCAGG GCAUAG(SEQ ID NO: 33) (SEQ ID NO: 133) 34 asiSRD5A1 34 UAUGCCCUGGCCAGCUAGCUGGCCAGGGCAU AGCCAC (SEQ ID NO: 34) (SEQ ID NO: 134) 35 asiSRD5A1 35GCUAUGCCCUGGCCAG CUGGCCAGGGCAUAG CCACAC (SEQ ID NO: 35) (SEQ ID NO: 135)36 asiSRD5A1 36 UCAUGAGUGGUACCUC GAGGUACCACUCAUG AUGCUC (SEQ ID NO: 36)(SEQ ID NO: 136) 37 asiSRD5A1 37 CAUCAUGAGUGGUACC GGUACCACUCAUGAU GCUCUU(SEQ ID NO: 37) (SEQ ID NO: 137) 38 asiSRD5A1 38 UCCGGAAAUUUGAAGAUCUUCAAAUUUCCGG AGGUAC (SEQ ID NO: 38) (SEQ ID NO: 138) 39 asiSRD5A1 39CAGUGUAUGCUGAUGA UCAUCAGCAUACACU GCACAA (SEQ ID NO: 39) (SEQ ID NO: 139)40 asiSRD5A1 40 GCAUGUUGAUAAACAU AUGUUUAUCAACAUG CCCGUU (SEQ ID NO: 40)(SEQ ID NO: 140) 41 asiSRD5A1 41 GUGGCUAUGCCCUGGC GCCAGGGCAUAGCCA CACCAC(SEQ ID NO: 41) (SEQ ID NO: 141) 42 asiSRD5A1 42 GUGUGGCUAUGCCCUGCAGGGCAUAGCCACA CCACUC (SEQ ID NO: 42) (SEQ ID NO: 142) 43 asiSRD5A1 43UGGUGUGGCUAUGCCC GGGCAUAGCCACACC ACUCCA (SEQ ID NO: 43) (SEQ ID NO: 143)44 asiSRD5A1 44 AGUGGUGUGGCUAUGC GCAUAGCCACACCAC UCCAUG (SEQ ID NO: 44)(SEQ ID NO: 144) 45 asiSRD5A1 45 UCUUCACGUUUUGUUU AAACAAAACGUGAAG AAAGCA(SEQ ID NO: 45) (SEQ ID NO: 145) 46 asiSRD5A1 46 GACUUGAGAACCCUUUAAAGGGUUCUCAAGU CAGGCU (SEQ ID NO: 46) (SEQ ID NO: 146) 47 asiSRD5A1 47CUGUUGGCGUGUACAA UUGUACACGCCAACA GUGGCA (SEQ ID NO: 47) (SEQ ID NO: 147)48 asiSRD5A1 48 UUAUUUGAAUACGUAA UUACGUAUUCAAAUA AGCCUC (SEQ ID NO: 48)(SEQ ID NO: 148) 49 asiSRD5A1 49 UUCCAAUGGCGCUUCU AGAAGCGCCAUUGGA AAGCUU(SEQ ID NO: 49) (SEQ ID NO: 149) 50 asiSRD5A1 50 AAAGGCAUCUGGACUUAAGUCCAGAUGCCUU UGCCUC (SEQ ID NO: 50) (SEQ ID NO: 150) 51 asiSRD5A1 51AUCAAUGUGCUCUGGU ACCAGAGCACAUUGA UGGCUC (SEQ ID NO: 51) (SEQ ID NO: 151)52 asiSRD5A1 52 GAUCACUUUCUGUAAC GUUACAGAAAGUGAU CAUUCU (SEQ ID NO: 52)(SEQ ID NO: 152) 53 asiSRD5A1 53 AUCUUCCUUCUAAUAG CUAUUAGAAGGAAGA UUAGCU(SEQ ID NO: 53) (SEQ ID NO: 153) 54 asiSRD5A1 54 GGCAUUGCUUUGCCUUAAGGCAAAGCAAUGC CAGAUG (SEQ ID NO: 54) (SEQ ID NO: 154) 55 asiSRD5A1 55UGUACAAUGGCGAUUA UAAUCGCCAUUGUAC ACGCCA (SEQ ID NO: 55) (SEQ ID NO: 155)56 asiSRD5A1 56 CUUCUCUAUGGACUUU AAAGUCCAUAGAGAA GCGCCA (SEQ ID NO: 56)(SEQ ID NO: 156) 57 asiSRD5A1 57 UUCCAAGGUGAGGCAA UUGCCUCACCUUGGA AGGGCC(SEQ ID NO: 57) (SEQ ID NO: 157) 58 asiSRD5A1 58 UCCAAGGUGAGGCAAAUUUGCCUCACCUUGG AAGGGC (SEQ ID NO: 58) (SEQ ID NO: 158) 59 asiSRD5A1 59GGUUCAUACGGAGUAA UUACUCCGUAUGAAC CACCAC (SEQ ID NO: 59) (SEQ ID NO: 159)60 asiSRD5A1 60 AUAGUAGAGAUUGUUG CAACAAUCUCUACUA UAUCCA (SEQ ID NO: 60)(SEQ ID NO: 160) 61 asiSRD5A1 61 UGUUGUCUGUGAAAUU AAUUUCACAGACAAC AAUCUC(SEQ ID NO: 61) (SEQ ID NO: 161) 62 asiSRD5A1 62 UUCAAGCUCUGGGUAAUUACCCAGAGCUUGA AAUUCU (SEQ ID NO: 62) (SEQ ID NO: 162) 63 asiSRD5A1 63UACCUAAUAAGUACCU AGGUACUUAUUAGGU AGAUUG (SEQ ID NO: 63) (SEQ ID NO: 163)64 asiSRD5A1 64 AUUGUUGUCUGUGAAA UUUCACAGACAACAA UCUCUA (SEQ ID NO: 64)(SEQ ID NO: 164) 65 asiSRD5A1 65 CAAAAGAGCAUCAUGA UCAUGAUGCUCUUUU GCUCUA(SEQ ID NO: 65) (SEQ ID NO: 165) 66 asiSRD5A1 66 CUAUGGACUUUGUAAAUUUACAAAGUCCAUA GAGAAG (SEQ ID NO: 66) (SEQ ID NO: 166) 67 asiSRD5A1 67CUGUCUUUGAUGGCAU AUGCCAUCAAAGACA GUUGUA (SEQ ID NO: 67) (SEQ ID NO: 167)68 asiSRD5A1 68 UCUACCUAAUAAGUAC GUACUUAUUAGGUAG AUUGCA (SEQ ID NO: 68)(SEQ ID NO: 168) 69 asiSRD5A1 69 CUAAUCUUCCUUCUAA UUAGAAGGAAGAUUA GCUAUG(SEQ ID NO: 69) (SEQ ID NO: 169) 70 asiSRD5A1 70 CAUUUUCAGAACAAUAUAUUGUUCUGAAAAU GCCAUC (SEQ ID NO: 70) (SEQ ID NO: 170) 71 asiSRD5A1 71GAUCUCUUCAAGGUCA UGACCUUGAAGAGAU CACUGU (SEQ ID NO: 71) (SEQ ID NO: 171)72 asiSRD5A1 72 AGAUUGUUGUCUGUGA UCACAGACAACAAUC UCUACU (SEQ ID NO: 72)(SEQ ID NO: 172) 73 asiSRD5A1 73 AGAGAUUGUUGUCUGU ACAGACAACAAUCUC UACUAU(SEQ ID NO: 73) (SEQ ID NO: 173) 74 asiSRD5A1 74 AGACGAACUCAGUGUAUACACUGAGUUCGUC UGACGA (SEQ ID NO: 74) (SEQ ID NO: 174) 75 asiSRD5A1 75UCCUCCUGGCCAUGUU AACAUGGCCAGGAGG AUGCAG (SEQ ID NO: 75) (SEQ ID NO: 175)76 asiSRD5A1 76 CUUAAUUUACCCAUUU AAAUGGGUAAAUUAA GCACCG (SEQ ID NO: 76)(SEQ ID NO: 176) 77 asiSRD5A1 77 UGAUGCGAGGAGGAAA UUUCCUCCUCGCAUC AGAAAU(SEQ ID NO: 77) (SEQ ID NO: 177) 78 asiSRD5A1 78 UGUUCUGUACCUGUAAUUACAGGUACAGAAC AUAAUC (SEQ ID NO: 78) (SEQ ID NO: 178) 79 asiSRD5A1 79CCUGUAACGGCUAUUU AAAUAGCCGUUACAG GUACAG (SEQ ID NO: 79) (SEQ ID NO: 179)80 asiSRD5A1 80 CCAUUGUGCAGUGUAU AUACACUGCACAAUG GCUCAA (SEQ ID NO: 80)(SEQ ID NO: 180) 81 asiSRD5A1 81 AACAUCCAUUCAGAUC GAUCUGAAUGGAUGU UUAUCA(SEQ ID NO: 81) (SEQ ID NO: 181) 82 asiSRD5A1 82 UAUCCAAAGUUCAGAAUUCUGAACUUUGGAU ACUCUU (SEQ ID NO: 82) (SEQ ID NO: 182) 83 asiSRD5A1 83ACCUAAAUACGCUGAA UUCAGCGUAUUUAGG UACUUA (SEQ ID NO: 83) (SEQ ID NO: 183)84 asiSRD5A1 84 CGCUGAAAUGGAGGUU AACCUCCAUUUCAGC GUAUUU (SEQ ID NO: 84)(SEQ ID NO: 184) 85 asiSRD5A1 85 AAUGGAGGUUGAAUAU AUAUUCAACCUCCAU UUCAGC(SEQ ID NO: 85) (SEQ ID NO: 185) 86 asiSRD5A1 86 AUAUCCUACUGUGUAAUUACACAGUAGGAUA UUCAAC (SEQ ID NO: 86) (SEQ ID NO: 186) 87 asiSRD5A1 87UAUGAGACUAGACUUU AAAGUCUAGUCUCAU ACACAC (SEQ ID NO: 87) (SEQ ID NO: 187)88 asiSRD5A1 88 AAUGUCACAAUCCCUU AAGGGAUUGUGACAU UUAUUG (SEQ ID NO: 88)(SEQ ID NO: 188) 89 asiSRD5A1 89 GGUCAACUGCAGUGUU AACACUGCAGUUGAC CUUGAA(SEQ ID NO: 89) (SEQ ID NO: 189) 90 asiSRD5A1 90 GCCAUUGUGCAGUCAUAUGACUGCACAAUGG CUACCC (SEQ ID NO: 90) (SEQ ID NO: 190) 91 asiSRD5A1 91UGUAAGUGGAGAACUU AAGUUCUCCACUUAC ACACAG (SEQ ID NO: 91) (SEQ ID NO: 191)92 asiSRD5A1 92 CUCUGCCUGUGUGAGU ACUCACACAGGCAGA GCAGCU (SEQ ID NO: 92)(SEQ ID NO: 192) 93 asiSRD5A1 93 ACCGUGAGCCAUCAAU AUUGAUGGCUCACGG UGAGUG(SEQ ID NO: 93) (SEQ ID NO: 193) 94 asiSRD5A1 94 GGUUUCUCUCUGUCUUAAGACAGAGAGAAAC CAUGUC (SEQ ID NO: 94) (SEQ ID NO: 194) 95 asiSRD5A1 95UAGUCUAGACCUAGUU AACUAGGUCUAGACU AGAAGA (SEQ ID NO: 95) (SEQ ID NO: 195)96 asiSRD5A1 96 UAGUGUAAAGAAUGAU AUCAUUCUUUACACU ACAAGG (SEQ ID NO: 96)(SEQ ID NO: 196) 97 asiSRD5A1 97 CUGUACCUGUUAUCAA UUGAUAACAGGUACA GGCUAU(SEQ ID NO: 97) (SEQ ID NO: 197) 98 asiSRD5A1 98 GAAUGCUUCAUGACUUAAGUCAUGAAGCAUU CAACAG (SEQ ID NO: 98) (SEQ ID NO: 198) 99 asiSRD5A1 99UGCCUUAUCAUCUCAU AUGAGAUGAUAAGGC AAAGCA (SEQ ID NO: 99) (SEQ ID NO: 199)100 asiSRD5A1 100 CAUCUCAUCUGGAGUU AACUCCAGAUGA GAUGAUAAG(SEQ ID NO: 100) (SEQ ID NO: 200)

[Example 2] Screening for RNAi-Inducing Double-Stranded Nucleic AcidMolecules Targeting SRD5A1

To confirm gene inhibitory efficiency at the mRNA level, 100 selectedkinds of asiRNA were transfected into a HuH-7 cell line at aconcentration of 0.3 nM, and qRT-PCR was performed to measure theexpression level of SRD5A1 mRNA.

The HuH-7 cell line was cultured in Dulbecco's Modified Eagle's Medium(DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100units/ml of penicillin 100 μg/ml of streptomycin. HuH-7 cells wereseeded in a 96-well plate at a density of 5×10³ cells/well, and areverse transfection experiment was conducted using asiRNA (0.3 nM, OliXInc.) and Lipofectamine 2000 (1 μl/ml, Invitrogen Inc.) in Opti-MEM (atotal volume of 100 μl) in accordance with Invitrogen's protocol. After24 hours, RNA purification and cDNA synthesis were performed inaccordance with a basic protocol provided by TOYOBO SuperPrep, theexpression level of the SRD5A1 gene was examined with a SRD5A1 TaqManprobe (Hs00602694_mH) using a Bio-Rad CFX-4000 machine, and the resultsare illustrated in FIGS. 1A and 1B.

From the results of screening the 100 kinds of asiRNA, the 12 top-rankedasiRNAs (in Table 1, No. 5, 6, 15, 18, 40, 48, 49, 59, 62, 77, and 86)were selected, followed by treatment with 0.3 nM, 1 nM, 3 nM, or 10 nMof asiRNA and western blotting to analyze the protein expressioninhibitory effects thereof. HuH-7 cells were seeded in a 6-well plate ata density of 2.5×10³ cells/well, and then a reverse transfectionexperiment was conducted using asiRNA and Lipofectamine 2000 (1 μl/ml,Invitrogen Inc.) in Opti-MEM (a total volume of 2 ml) in accordance withInvitrogen's protocol. After 48 hours, the cells were lysed using amammalian protein extraction buffer (GE healthcare), and then proteinswere quantified using a Bradford assay. 10 μg of the protein of eachsample was electrophoresed using 12% SDS-PAGE at 80 V for 20 minutes andat 120 V for 1 hour, and then transferred onto a PVDE membrane (Bio-Rad)at 300 mA for 1 hour and 20 minutes. After transfer, the membrane wasblocked in 5% skim milk for 1 hour and allowed to react with SRD5A1antibody (ABcam, ab110123) at a ratio of 1:2000 for 12 hours. The nextday, the resulting membrane was allowed to react with anti-Goat HRP(Santa Cruz) at a ratio of 1:10000 for 1 hour, and then the expressionlevels of the SRD5A1 protein were compared with each other usingChemiDoc (Bio-Rad). From the results of FIG. 2, 4 asiRNAs (in Table 1,No. 48, 49, 69, and 86) capable of inhibiting SRD5A1 protein expressionwere selected.

[Example 3] 16 Kinds of Cp-asiRNA Targeting SRD5A1 Gene and Having SelfCell-Penetrating Ability

SRD5A1 cp-asiRNAs (a total of 16 strands) were designed by applyingthree modification patterns to 4 kinds (in Table 1, No. 48, 49, 69, and86) of asiRNA targeting SRD5A1 according to the number and position of2′OMe (methyl), phosphorothioate bonds (PS), and cholesterol, and thensynthesized by OliX Inc. (Korea). cp-asiRNA enhances endocytosisefficiency and stability and thus may penetrate through the cellmembrane with high efficiency without the aid of a delivery vehicle tothereby inhibit target gene expression. The synthesized sense andantisense strand RNA oligonucleotides were annealed at 95° C. for 2minutes through incubation at 37° C. for 1 hour, and cp-asiRNAs annealedby 10% polyacrylamide gel electrophoresis (PAGE) were confirmed by a UVtransilluminator.

TABLE 2 16 strands of cp-asiRNA nucleotide sequences targeting SRD5A1No. Name Sequence (5′-->3′)  1 SRD5A1cp- mUUmAUmUUmGAmAUmACmGU*mA*A*asiRNA S  cholesterol 48  2 SRD5A1cp- UUACGUAUUCAAAUmAmAG*C*C*U*CasiRNA AS  48(2, 4)  3 SRD5A1cp- UUACGUAUUCAAAUmAmAmG*mC*C*U*CasiRNA AS  48(4, 4)  4 SRD5A1cp- UUACGUAUUCAAAUmAmAmG*mC*mC*mU*mCasiRNA AS  48(7, 4)  5 SRD5A1cp- mUUmCCmAAmUGmGCmGCmUU*mC*U* asiRNA S cholesterol 49  6 SRD5A1cp- AGAAGCGCCAUUGGmAmAA*G*C*U*U asiRNA AS 49(2, 4)  7 SRD5A1cp- AGAAGCGCCAUUGGmAmAmA*mG*C*U*U asiRNA AS  49(4, 4) 8 SRD5A1cp- AGAAGCGCCAUUGGmAmAmA*mG*mC*mU*mU asiRNA AS  49(7, 4)  9SRD5A1cp- mCUmAAmUCmUUmCCmUUmCU*mA*A* asiRNA S  cholesterol 69 10SRD5A1cp- UUAGAAGGAAGAUUmAmGC*U*A*U*G asiRNA AS  69(2, 4) 11 SRD5A1cp-UUAGAAGGAAGAUUmAmGmC*mU*A*U*G asiRNA AS  69(4, 4) 12 SRD5A1cp-UUAGAAGGAAGAUUmAmGmC*mU*mA*mU*mG asiRNA AS  69(7, 4) 13 SRD5A1cp-mAUmAUmCCmUAmCUmGUmGU*mA*A* asiRNA S  cholesterol 86 14 SRD5A1cp-UUACACAGUAGGAUmAmUU*C*A*A*C asiRNA AS  86(2, 4) 15 SRD5A1cp-UUACACAGUAGGAUmAmUmU*mC*A*A*C asiRNA AS  86(4, 4) 16 SRD5A1cp-UUACACAGUAGGAUmAmUmU*mC*mA*mA*mC asiRNA AS  86(7, 4) m: 2′-O-Methyl/RNA*: phosphorothioated bond

[Example 4] Screening for Cp-asiRNA Targeting SRD5A1 Gene and HavingSelf Cell-Penetrating Ability

The inhibitory effects of the 12 kinds of cp-asiRNA shown in Table 2against SRD5A1 expression were examined. A HuH-7 cell line was incubatedwith 1 μM or 3 μM of 12 kinds of cp-asiRNA in Opti-MEM media for 24hours, and then the media were replaced with Dulbecco's Modified Eagle'sMedium (Gibco) containing 10% fetal bovine serum (Gibco) and 100units/ml penicillin 100 μg/ml streptomycin, and 24 hours after mediareplacement, SRD5A1 expression was examined at the protein level. Asillustrated in FIG. 3, as the result of repeatedly conducting twoexperiments, it was confirmed that SRD5A1 cp-asiRNA #49(2,4) and#86(7,4) exhibited gene inhibitory efficiency of 50% or higher.

The inhibitory effects of the two selected kinds of cp-asiRNA againstSRD5A1 expression were examined in a HuH-7 cell line. The HuH-7 cellline was incubated with 0.3 μM, 1 μM, or 3 μM of each of the two kindsof cp-asiRNA in Opti-MEM media for 24 hours, and then SRD5A1 expressionwas examined at the mRNA level. As the result of repeatedly conductingfour experiments, it was confirmed that SRD5A1 cp-asiRNA #49(2,4) and#86(7,4) exhibited gene inhibitory efficiency of 50% or higher at aconcentration of 1 μM or higher (see FIG. 4).

[Example 5] Screening for 112 Kinds of RNAi-Inducing Double-StrandedNucleic Acid Molecules Targeting SRD5A2

To obtain high-efficiency RNAi-inducing double-stranded nucleic acidmolecules targeting SRD5A2, the target sequence of the SRD5A2 gene wasselected and then asiRNA was designed. The asiRNA structure is differentfrom that of generally known siRNAs, and thus when the nucleotidesequences of asiRNA are designed using a general siRNA design program,it may be somewhat difficult to design an optimized asiRNA. Therefore,asiRNA was constructed by the following method. An NCBI db search wasused to obtain information on the SRD5A2 gene (mRNA Accession Number:NM_000348.3) which is thought to target male pattern hair loss(androgenetic hair loss). For subsequent animal experiments, nucleotidesequences with at least 80% homology to that of mice were secured, andthen 100 kinds of asiRNA were designed according to a design method suchas the exclusion of sequences having a GC content of 30-62% and 4 ormore G or C consecutive bases, and then synthesized by OliX Inc.(Korea). The synthesized sense and antisense strand RNA oligonucleotideswere annealed at 95° C. for 2 minutes through incubation at 37° C. for 1hour, and the asiRNA annealed by 10% polyacrylamide gel electrophoresis(PAGE) was confirmed using a UV transilluminator.

TABLE 3 112 strands of asiRNA nucleotide sequencestargeting 3-oxo-5-alpha-steroid 4- dehydrogenase 2 112 kindsSequence (5′-3′) No. Name S AS   1 asiSRD5A2  GUUCCUGCAGGAGCUGCAGCUCCUGCAGGAACCAGGC 1 (SEQ ID NO: 201) (SEQ ID NO: 313)   2 asiSRD5A2 UUCCUGCAGGAGCUGC GCAGCUCCUGCAGGAACCAGG 2 (SEQ ID NO: 202)(SEQ ID NO: 314)   3 asiSRD5A2  UCCUGCAGGASCUGCC GGCAGCUCCUGCAGGAACCAG 3(SEQ ID NO: 203) (SEQ ID NO: 315)   4 asiSRD5A2  CCUGCAGGAGCUGCCUAGGCAGCUCCUGCAGGAACCA 4 (SEQ ID NO: 204) (SEQ ID NO: 316)   5 asiSRD5A2 CUGCAGGAGCUGCCUU AAGGCAGCUCCUGCAGGAACC 5 (SEQ ID NO: 205)(SEQ ID NO: 317)   6 asiSRD5A2  UGCAGGAGCUGCCUUC GAAGGCAGCUCCUGCAGGAAC 6(SEQ ID NO: 206) (SEQ ID NO: 318)   7 asiSRD5A2  GCAGGAGCUGCCUUCCGGAAGGCAGCUCCUGCAGGAA 7 (SEQ ID NO: 207) (SEQ ID NO: 319)   8 asiSRD5A2 CAGGAGCUGCCUUCCU AGGAAGGCAGCUCCUGCAGGA 8 (SEQ ID NO: 208)(SEQ ID NO: 320)   9 asiSRD5A2  AGGAGCUGCCUUCCUU AAGGAAGGCAGCUCCUGCAGG 9(SEQ ID NO: 209) (SEQ ID NO: 321)  10 asiSRD5A2  ACUUCCACAGGACAUUAAUGUCCUGUGGAAGUAAUGU 10 (SEQ ID NO: 210) (SEQ ID NO: 322)  11asiSRD5A2  CUUCCACAGGACAUUU AAAUGUCCUGUGGAAGUAAUG 11 (SEQ ID NO: 211)(SEQ ID NO: 323)  12 asiSRD5A2  AGGUGGCUUGUUUACG CGUAAACAAGCCACCUUGUGG12 (SEQ ID NO: 212) (SEQ ID NO: 324)  13 asiSRD5A2  GGUGGCUUGUUUACGUACGUAAACAAGCCACCUUGUG 13 (SEQ ID NO: 213) (SEQ ID NO: 325)  14asiSRD5A2  GUGGCUUGUUUACGUA UACGUAAACAAGCCACCUUGU 14 (SEQ ID NO: 214)(SEQ ID NO: 326)  15 asiSRD5A2  UGGCUUGUUUACGUAU AUACGUAAACAAGCCACCUUG15 (SEQ ID NO: 215) (SEQ ID NO: 327)  16 asiSRD5A2  GGCUUGUUUACGUAUGCAUACGUAAACAAGCCACCUU 16 (SEQ ID NO: 216) (SEQ ID NO: 328)  17asiSRD5A2  GCUUGUUUACGUAUGU ACAUACGUAAACAAGCCACCU 17 (SEQ ID NO: 217)(SEQ ID NO: 329)  18 asiSRD5A2  CUACCUCAAGAUGUUU AAACAUCUUGAGGUAGAACCU18 (SEQ ID NO: 218) (SEQ ID NO: 330)  19 asiSRD5A2  GGUUCCUGCAGGAGCUAGCUCCUGCAGGAACCAGGCG 19 (SEQ ID NO: 219) (SEQ ID NO: 331)  20asiSRD5A2  UACUUCCACAGGACAU AUGUCCUGUGGAAGUAAUGUA 20 (SEQ ID NO: 220)(SEQ ID NO: 332)  21 asiSRD5A2  AGACAUACGGUUUAGC GCUAAACCGUAUGUCUGUGUA21 (SEQ ID NO: 221) (SEQ ID NO: 333)  22 asiSRD5A2  CAGACAUACGGUUUAGCUAAACCGUAUGUCUGUGUAC 22 (SEQ ID NO: 222) (SEQ ID NO: 334)  23asiSRD5A2  ACAGACAUACGGUUUA UAAACCGUAUGUCUGUGUACC 23 (SEQ ID NO: 223)(SEQ ID NO: 335)  24 asiSRD5A2  CACAGACAUACGGUUU AAACCGUAUGUCUGUGUACCA24 (SEQ ID NO: 224) (SEQ ID NO: 336)  25 asiSRD5A2  GGAUUCCACAAGGUGGCCACCUUGUGGAAUCCUGUAG 25 (SEQ ID NO: 225) (SEQ ID NO: 337)  26asiSRD5A2  GAUUCCACAAGGUGGC GCCACCUUGUGGAAUCCUGUA 26 (SEQ ID NO: 226)(SEQ ID NO: 338)  27 asiSRD5A2  AUUCCACAAGGUGGCU AGCCACCUUGUGGAAUCCUGU27 (SEQ ID NO: 227) (SEQ ID NO: 339)  28 asiSRD5A2  UUCCACAAGGUGGCUUAAGCCACCUUGUGGAAUCCUG 28 (SEQ ID NO: 228) (SEQ ID NO: 340)  29asiSRD5A2  UCCACAAGGUGGCUUG CAAGCCACCUUGUGGAAUCCU 29 (SEQ ID NO: 228)(SEQ ID NO: 341)  30 asiSRD5A2  CCACAAGGUGGCUUGU ACAAGCCACCUUGUGGAAUCC30 (SEQ ID NO: 230) (SEQ ID NO: 342)  31 asiSRD5A2  CACAAGGUGGCUUGUUAACAAGCCACCUUGUGGAAUC 31 (SEQ ID NO: 231) (SEQ ID NO: 343)  32asiSRD5A2  ACAAGGUGGCUUGUUU AAACAAGCCACCUUGUGGAAU 32 (SEQ ID NO: 232)(SEQ ID NO: 344)  33 asiSRD5A2  CAAGGUGGCUUGUUUA UAAACAAGCCACCUUGUGGAA33 (SEQ ID NO: 233) (SEQ ID NO: 345)  34 asiSRD5A2  AAGGUGGCUUGUUUACGUAAACAAGCCACCUUGUGGA 34 (SEQ ID NO: 234) (SEQ ID NO: 346)  35asiSRD5A2  CUGGAGCCAAUUUCCU AGGAAAUUGGCUCCAGAAACA 35 (SEQ ID NO: 235)(SEQ ID NO: 347)  36 asiSRD5A2  UCUGGAGCCAAUUUCC GGAAAUUGGCUCCAGAAACAU36 (SEQ ID NO: 236) (SEQ ID NO: 348)  37 asiSRD5A2  UUCUGGAGCCAAUUUCGAAAUUGGCUCCAGAAACAUA 37 (SEQ ID NO: 237) (SEQ ID NO: 349)  38asiSRD5A2  UUUCUGGAGCCAAUUU AAAUUGGCUCCAGAAACAUAC 38 (SEQ ID NO: 238)(SEQ ID NO: 350)  39 asiSRD5A2  GUUUCUGGAGCCAAUU AAUUGGCUCCAGAAACAUACG39 (SEQ ID NO: 239) (SEQ ID NO: 351)  40 asiSRD5A2  UGUUUCUGGAGCCAAUAUUGGCUCCAGAAACAUACGU 40 (SEQ ID NO: 240) (SEQ ID NO: 352)  41asiSRD5A2  AUGUUUCUGGAGCCAA UUGGCUCCAGAAACAUACGUA 41 (SEQ ID NO: 241)(SEQ ID NO: 353)  42 asiSRD5A2  UAUGUUUCUGGAGCCA UGGCUCCAGAAACAUACGUAA42 (SEQ ID NO: 242) (SEQ ID NO: 354)  43 asiSRD5A2  GUAUGUUUCUGGAGCCGGCUCCAGAAACAUACGUAAA 43 (SEQ ID NO: 243) (SEQ ID NO: 355)  44asiSRD5A2  GCUCCAGAAACAUACG GCUCCAGAAACAUACGUAAAC 44 (SEQ ID NO: 244)(SEQ ID NO: 356)  45 asiSRD5A2  CAUAGGUUCUACCUCA UGAGGUAGAACCUAUGGUCGU45 (SEQ ID NO: 245) (SEQ ID NO: 357)  46 asiSRD5A2  AUAGGUUCUACCUCAAUUGAGGUAGAACCUAUGGUGG 46 (SEQ ID NO: 246) (SEQ ID NO: 358)  47asiSRD5A2  UAGGUUCUACCUCAAG CUUGAGGUAGAACCUAUGGUG 47 (SEQ ID NO: 247)(SEQ ID NO: 359)  48 asiSRD5A2  AGGUUCUACCUCAAGA UCUUGAGGUAGAACCUAUGGU48 (SEQ ID NO: 248) (SEQ ID NO: 360)  49 asiSRD5A2  GGUUCUACCUCAAGAUAUCUUGAGGUAGAACCUAUGG 49 (SEQ ID NO: 249) (SEQ ID NO: 361)  50asiSRD5A2  GUUCUACCUCAAGAUG CAUCUUGAGGUAGAACCUAUG 50 (SEQ ID NO: 250)(SEQ ID NO: 362)  51 asiSRD5A2  UUCUACCUCAAGAUGU ACAUCUUGAGGUAGAACCUAU51 (SEQ ID NO: 251) (SEQ ID NO: 363)  52 asiSRD5A2  UCUACCUCAAGAUGUUAACAUCUUGAGGUAGAACCUA 52 (SEQ ID NO: 252) (SEQ ID NO: 364)  53asiSRD5A2  CAAAUCUCGGAAAGCC GGCUUUCCGAGAUUUGGGGUA 53 (SEQ ID NO: 253)(SEQ ID NO: 365)  54 asiSRD5A2  AAAUCUCGGAAAGCCC GGGCUUUCCGAGAUUUGGGGU54 (SEQ ID NO: 254) (SEQ ID NO: 366)  55 asiSRD5A2  AAUCUCGGAAAGCCCUAGGGCUUUCCGAGAUUUGGGG 55 (SEQ ID NO: 255) (SEQ ID NO: 367)  56asiSRD5A2  GCCCUUAUUCCAUUCA UGAAUGGAAUAAGGGCUUUCC 56 (SEQ ID NO: 256)(SEQ ID NO: 368)  57 asiSRD5A2 CCCUUAUUCCAUUCAU AUGAAUGGAAUAAGGGCUUUC 57(SEQ ID NO: 257) (SEQ ID NO: 369)  58 asiSRD5A2  CCUUAUUCCAUUCAUCGAUGAAUGGAAUAAGGGCUUU 58 (SEQ ID NO: 258) (SEQ ID NO: 370)  59asiSRD5A2  CUUAUUCCAUUCAUCU AGAUGAAUGGAAUAAGGGCUU 59 (SEQ ID NO: 259)(SEQ ID NO: 371)  60 asiSRD5A2  UUAUUCCAUUCAUCUU AAGAUGAAUGGAAUAAGGGCU60 (SEQ ID NO: 260) (SEQ ID NO: 372)  61 asiSRD5A2  UAUUCCAUUCAUCUUUAAAGAUGAAUGGAAUAAGGGC 61 (SEQ ID NO: 261) (SEQ ID NO: 373)  62asiSRD5A2  AUUCCAUUCAUCUUUU AAAAGAUGAAUGGAAUAAGGG 62 (SEQ ID NO: 262)(SEQ ID NO: 374)  63 asiSRD5A2  UUCCAUUCAUCUUUUA UAAAAGAUGAAUGGAAUAAGG63 (SEQ ID NO: 263) (SEQ ID NO: 375)  64 asiSRD5A2  UCCAUUCAUCUUUUAAUUAAAAGAUGAAUGGAAUAAG 64 (SEQ ID NO: 264) (SEQ ID NO: 376)  65asiSRD5A2  UCUCACUUUGUUUCCU AGGAAACAAAGUGAGAAAAAU 65 (SEQ ID NO: 265)(SEQ ID NO: 377)  66 asiSRD5A2  UUCUCACUUUGUUUCC GGAAACAAAGUGAGAAAAAUG66 (SEQ ID NO: 266) (SEQ ID NO: 378)  67 asiSRD5A2  UUUCUCACUUUGUUUCGAAACAAAGUGAGAAAAAUGC 67 (SEQ ID NO: 267) (SEQ ID NO: 379)  68asiSRD5A2  UUUUCUCACUUUGUUU AAACAAAGUGAGAAAAAUGCA 68 (SEQ ID NO: 268)(SEQ ID NO: 380)  69 asiSRD5A2  UUUUUCUCACUUUGUU AACAAAGUGAGAAAAAUGCAA69 (SEQ ID NO: 269) (SEQ ID NO: 381)  70 asiSRD5A2  AUUUUUCUCACUUUGUACAAAGUGAGAAAAAUGCAAA 70 (SEQ ID NO: 270) (SEQ ID NO: 382)  71asiSRD5A2  UGGCAGGCAGCGCCAC GUGGCGCUGCCUGCCAGCACU 71 (SEQ ID NO: 271)(SEQ ID NO: 363)  72 asiSRD5A2  CUGGCAGGCAGCGCCA UGGCGCUGCCUGCCAGCACUG72 (SEQ ID NO: 272) (SEQ ID NO: 384)  73 asiSRD5A2  GGCAGGCAGCGCCACUAGUGGCGCUGCCUGCCAGCAC 73 (SEQ ID NO: 273) (SEQ ID NO: 385)  74asiSRD5A2  UGCCAGCCCGCGCCGC GCGGCGCGGGCUGGCAGGCGG 74 (SEQ ID NO: 274)(SEQ ID NO: 386)  75 asiSRD5A2  UUACUUCCACAGGACA UGUCCUGUGGAAGUAAUGUAG75 (SEQ ID NO: 275) (SEQ ID NO: 387)  76 asiSRD5A2  GUGGAAGUAAUGUAGGCCUACAUUACUUCCACAGGAC 76 (SEQ ID NO: 276) (SEQ ID NO: 388)  77asiSRD5A2  CCCUGAUGGGUGGUAC GUACCACCCAUCAGGGUAUUC 77 (SEQ ID NO: 277)(SEQ ID NO: 389)  78 asiSRD5A2  CCUGAUGGGUGGUACA UGUACCACCCAUCAGGGUAUU78 (SEQ ID NO: 278) (SEQ ID NO: 390)  79 asiSRD5A2  CUGAUGGGUGGUACACGUGUACCACCCAUCAGGGUAU 79 (SEQ ID NO: 279) (SEQ ID NO: 391)  80asiSRD5A2  UGAUGGGUGGUACACA UGUGUACCACCCAUCAGGGUA 80 (SEQ ID NO: 280)(SEQ ID NO: 392)  81 asiSRD5A2  GAUGGGUGGUACACAG CUGUGUACCACCCAUCAGGGU81 (SEQ ID NO: 281) (SEQ ID NO: 393)  82 asiSRD5A2  AUGGGUGGUACACAGAUCUGUGUACCACCCAUCAGGG 82 (SEQ ID NO: 282) (SEQ ID NO: 394)  83asiSRD5A2  UGGGUGGUACACAGAC GUCUGUGUACCACCCAUCAGG 83 (SEQ ID NO: 283)(SEQ ID NO: 395)  84 asiSRD5A2  GGGUGGUACACAGACA UGUCUGUGUACCACCCAUCAG84 (SEQ ID NO: 284) (SEQ ID NO: 396)  85 asiSRD5A2  GGUGGUACACAGACAUAUGUCUGUGUACCACCCAUCA 85 (SEQ ID NO: 285) (SEQ ID NO: 397)  86asiSRD5A2  GACAUACGGUUUAGCU AGCUAAACCGUAUGUCUGUGU 86 (SEQ ID NO: 286)(SEQ ID NO: 390)  87 asiSRD5A2  CUUGGGUGUCUUCUUA UAAGAAGACACCCAAGCUAAA87 (SEQ ID NO: 287) (SEQ ID NO: 399)  88 asiSRD5A2  GCUUGGGUGUCUUCUUAAGAAGACACCCAAGCUAAAC 88 (SEQ ID NO: 288) (SEQ ID NO: 400)  89asiSRD5A2  AGCUUGGGUGUCUUCU AGAAGACACCCAAGCUAAACC 89 (SEQ ID NO: 289)(SEQ ID NO: 401)  90 asiSRD5A2  UAGCUUGGGUGUCUUC GAAGACACCCAAGCUAAACCG90 (SEQ ID NO: 290) (SEQ ID NO: 402)  91 asiSRD5A2  GCCAGCUCAGGAAGCCGGCUUCCUGAGCUGGCGCAAU 91 (SEQ ID NO: 291) (SEQ ID NO: 403)  92asiSRD5A2  CGCCAGCUCAGGAAGC GCUUCCUGAGCUGGCGCAAUA 92 (SEQ ID NO: 292)(SEQ ID NO: 404)  93 asiSRD5A2  GCGCCAGCUCAGGAAG CUUCCUGAGCUGGCGCAAUAU93 (SEQ ID NO: 293) (SEQ ID NO: 405)  94 asiSRD5A2  UGGAGCCAAUUUCCUCGAGGAAAUUGGCUCCAGAAAC 94 (SEQ ID NO: 294) (SEQ ID NO: 406)  95asiSRD5A2  CUCACUUUGUUUCCUU AAGGAAACAAAGUGAGAAAAA 95 (SEQ ID NO: 295)(SEQ ID NO: 407)  96 asiSRD5A2  CAUUUUUCUCACUUUG CAAAGUGAGAAAAAUGCAAAU96 (SEQ ID NO: 296) (SEQ ID NO: 408)  97 asiSRD5A2  CCAUAGGUUCUACCUCGAGGUAGAACCUAUGGUGGUG 97 (SEQ ID NO: 297) (SEQ ID NO: 409)  98asiSRD5A2  ACCAUAGGUUCUACCU AGGUAGAACCUAUGGUGGUGA 98 (SEQ ID NO: 298)(SEQ ID NO: 410)  99 asiSRD5A2  CACCAUAGGUUCUACC GGUAGAACCUAUGGUGGUGAA99 (SEQ ID NO: 299) (SEQ ID NO: 411) 100 asiSRD5A2  CCACCAUAGGUUCUACGUAGAACCUAUGGUGGUGAAA 100 (SEQ ID NO: 300) (SEQ ID NO: 412) 101asiSRD5A2  ACCACCAUAGGUUCUA UAGAACCUAUGGUGGUGAAAA 101 (SEQ ID NO: 301)(SEQ ID NO: 413) 102 asiSRD5A2  CACCACCAUAGGUUCU AGAACCUAUGGUGGUGAAAAG102 (SEQ ID NO: 302) (SEQ ID NO: 414) 103 asiSRD5A2  GGACUACCCCAAAUCUAGAUUUGGGGUAGUCCUCAAA 103 (SEQ ID NO: 303) (SEQ ID NO: 415) 104asiSRD5A2  AGGACUACCCCAAAUC GAUUUGGGGUAGUCCUCAAAC 104 (SEQ ID NO: 304)(SEQ ID NO: 416) 105 asiSRD5A2  GAGGACUACCCCAAAU AUUUGGGGUAGUCCUCAAACA105 (SEQ ID NO: 305) (SEQ ID NO: 417) 106 asiSRD5A2  UGAGGACUACCCCAAAUUUGGGGUAGUCCUCAAACAU 106 (SEQ ID NO: 306) (SEQ ID NO: 418) 107asiSRD5A2  UUGAGGACUACCCCAA UUGGGGUAGUCCUCAAACAUC 107 (SEQ ID NO: 307)(SEQ ID NO: 419) 108 asiSRD5A2  UUUGAGGACUACCCCA UGGGGUAGUCCUCAAACAUCU108 (SEQ ID NO: 308) (SEQ ID NO: 420) 109 asiSRD5A2  CCAAAUCUCGGAAAGCGCUUUCCGAGAUUUGGGGUAG 109 (SEQ ID NO: 309) (SEQ ID NO: 421) 110asiSRD5A2  AGCCCUUAUUCCAUUC GAAUGGAAUAAGGGCUUUCCG 110 (SEQ ID NO: 310)(SEQ ID NO: 422) 111 asiSRD5A2  AAGCCCUUAUUCCAUU AAUGGAAUAAGGGCUUUCCGA111 (SEQ ID NO: 311) (SEQ ID NO: 423) 112 asiSRD5A2  GGCUAUGCCCUGGCCAUGGCCAGGGCAUAGCCGAUCC 112 (SEQ ID NO: 312) (SEQ ID NO: 424)

[Example 6] Screening for RNAi-Inducing Double-Stranded Nucleic AcidMolecules Targeting SRD5A2

To confirm gene inhibitory efficiency at the mRNA level, 112 selectedkinds of asiRNA were transfected into a HuH-7 cell line at aconcentration of 0.3 nM, and qRT-PCR was performed to measure theexpression level of SRD5A1 mRNA. The HuH-7 cell line was cultured inDulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% fetalbovine serum (FBS, Gibco) and 100 units/ml of penicillin 100 μg/ml ofstreptomycin. HuH-7 cells were seeded in a 24-well plate at a density of5×10⁴ cells/well, and a reverse transfection experiment was conductedusing asiRNA (0.3 nM, OliX Inc.) and Lipofectamine 2000 (1 μl/ml,Invitrogen Inc.) in Opti-MEM (a total volume of 500 μl) in accordancewith Invitrogen's protocol. After 24 hours, total RNA was extractedusing TRIzol (TaKaPa), and then cDNA was synthesized using ahigh-capacity cDNA reverse transcription kit (Applied Biosystems), andthe expression level of the SRD5A2 gene was examined using power SYBRgreen PCR master Mix (Applied Biosystems), the following primers, and aStepOne real-time PCR system (see FIGS. 5A and 5B).

The nucleotide sequences of the primers used in the experiment are shownin Table 4 below.

TABLE 4 Primer nucleotide sequences Name Sequence (5′-3′) size HumanForward GAG TCA ACG GAT TTG GTC GT 186 GAPDH (SEQ ID NO: 425) ReverseGAC AAG CTT CCC GTT CTC AG (SEQ ID NO: 426) Human ForwardTGA ACC TGG GTG GCT TAT GA 242 SRD5A2 (SEQ ID NO: 427) ReverseGAA AGG AAA GTT GCT TGG G (SEQ ID NO: 428)

From the results of screening 112 asiRNAs, the 23 top-ranked asiRNAs (inTable 3, No. 5, 8, 28, 31, 32, 33, 37, 38, 39, 40, 42, 48, 49, 59, 60,62, 65, 83, 84, 85, 91, 92, and 100) were selected, and western blottingwas performed at a concentration of 10 nM. HuH-7 cells were seeded in a6-well plate at a density of 2.5×10³ cells/well, and then a reversetransfection experiment was conducted using asiRNA and Lipofectamine2000 (1 μl/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 2 ml) inaccordance with Invitrogen's protocol. After 48 hours, the cells werelysed using a mammalian protein extraction buffer (GE healthcare), andthen proteins were quantified using a Bradford assay. 10 μg of theprotein of each sample was electrophoresed using 12% SDS-PAGE at 80 Vfor 20 minutes and at 120 V for 1 hour, and then transferred onto a PVDEmembrane (Bio-Rad) at 300 mA for 1 hour and 20 minutes. After transfer,the membrane was blocked in 5% skim milk for 1 hour and then allowed toreact with SRD5A2 antibody (ABcam, ab124877) at a ratio of 1:2000 for 12hours. The next day, the resulting membrane was allowed to react withanti-Rabbit HRP (Santa Cruz) at a ratio of 1:10000 for 1 hour, and thenthe expression levels of the SRD5A2 protein were compared with eachother using ChemiDoc (Bio-Rad). In the present experiment, 4 asiRNAs (inTable 3, No. 31, 59, 60, and 62) capable of inhibiting SRD5A2 proteinexpression by about 50% or higher were selected (see FIGS. 6A and 6B).

[Example 7] 16 Kinds of Cp-asiRNA Targeting SRD5A2 Gene and Having SelfCell-Penetrating Ability

SRD5A2 cp-asiRNAs (total 16 strands) were designed by applying threemodification patterns to 4 kinds (in Table 3, No. 31, 59, 60, and 62) ofasiRNA targeting SRD5A2 according to the number and position of 2′OMe(methyl), phosphorothioate bonds (PS), and cholesterol, and thensynthesized by OliX Inc. (Korea). cp-asiRNA enhances endocytosisefficiency and stability and thus may penetrate through the cellmembrane with high efficiency without the aid of a delivery vehicle tothereby inhibit the expression of the target gene. The synthesized senseand antisense strand RNA oligonucleotides were annealed at 95° C. for 2minutes through incubation at 37° C. for 1 hour, and cp-asiRNAs annealedby 10% polyacrylamide gel electrophoresis (PAGE) were confirmed using aUV transilluminator.

TABLE 5 16 strands of cp-asiRNA nucleotide sequences targeting SRD5A2No. Name Sequence (5′-->3′)  1 SRD5A2cp- mCAmCAmAGmGUmGCmCUmU*G*mU*UasiRNA cholesterol S 31  2 SRD5A2cp- AACAAGCCACCUUGmUmGG*A*A*U*C asiRNAAS 31(2, 4)  3 SRD5A2cp- AACAAGCCACCUUGmUmSmG*mA*A*U*C asiRNAAS 31(4, 4)  4 SRD5A2cp- AACAAGCCACCUUGmUmGmG*mA*mA*mU*mC asiRNAAS 31(7, 4)  5 SRD5A2cp- mCUmUAmUUmCCmAUMUCmA*U*mC*U asiRNA cholesterolS 59  6 SRD5A2cp- AGAUGAAUGGAAUAmAmGG*G*C*U*U asiRNA AS 59(2, 4)  7SRD5A2cp- AGAUGAAUGGAAUAmAmGmG*mG*C*U*U asiRNA AS 59(4, 4)  8 SRD5A2cp-AGAUGAAUGGAAUAmAmGmG*mG*mC*mU*mU asiRNA AS 59(7, 4)  9 SRD5A2cp-mUUmAUmUCmCAmUUmCAmU*C*mU*U asiRNA cholesterol S 60 10 SRD5A2cp-AAGAUGAAUGGAAUmAmAG*G*G*C*U asiRNA AS 60(2, 4) 11 SRD5A2cp-AAGAUGAAUGGAAUmAmAmG*mG*G*C*U asiRNA AS 60(4, 4) 12 SRD5A2cp-AAGAUGAAUGGAAUmAmAmG*mG*mG*mC*mU asiRNA AS 60(7, 4) 13 SRD5A2cp-mAUmUCmCAmUUmCAmUC*mU*U*mU*U asiRNA cholesterol S 62 14 SRD5A2cp-AAAAGAUGAAUGGAmAmUA*A*G*G*G asiRNA AS 62(2, 4) 15 SRD5A2cp-AAAAGAUGAAUGGAmAmUmA*mA*G*G*G asiRNA AS 62(4, 4) 16 SRD5A2cp-AAAAGAUGAAUGGAmAmUmA*mA*mG*mG*mG asiRNA AS 62(7, 4) m: 2′-O-Methyl/RNA*: phosphorothioated bond

[Example 8] Screening for Cp-asiRNA Targeting SRD5A2 Gene and HavingSelf Cell-Penetrating Ability

The inhibitory effects of the 12 kinds of cp-asiRNA shown in Table 5against SRD5A2 expression were examined. A HuH-7 cell line was incubatedwith 1 μM or 3 μM of 12 kinds of cp-asiRNA in Opti-MEM media for 24hours, and then the media were replaced with Dulbecco's Modified Eagle'sMedium (Gibco) containing 10% fetal bovine serum (Gibco) and 100units/ml penicillin 100 μg/ml streptomycin, and 24 hours after mediareplacement, SRD5A2 expression was examined at the protein level. As theresult of repeatedly conducting two experiments, it was confirmed thatSRD5A2 cp-asiRNA #59(4,4) and #62(4,4) exhibited gene inhibitoryefficiency of 50% or higher (see FIG. 7).

[Example 9] Confirmation of Inhibitory Efficiency of 2 Selected Kinds ofCp-asiRNA Against Target Gene SRD5A2 Expression

The inhibitory effects of the two above-selected kinds of cp-asiRNAagainst SRD5A2 expression were examined in a HuH-7 cell line. The HuH-7cell line was incubated in Opti-MEM media with 1.95 nM, 3.9 nM, 7.8 nM,15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, or 1,000 nM of eachof the two kinds of cp-asiRNA for 24 hours, and then SRD5A2 expressionwas examined at the mRNA level. As the result of repeatedly conductingthree experiments, it was confirmed that SRD5A2 cp-asiRNA #59(4,4) and#62(4,4) had IC₅₀ values of 22.37 nM and 27.18 nM, respectively (seeFIG. 8).

The inhibitory effects of the two above-selected kinds of cp-asiRNAagainst SRD5A2 expression were examined in a HuH-7 cell line. The HuH-7cell line was incubated with 0.1 μM, 0.3 μM, 1 μM, or 3 μM of the twokinds of cp-asiRNA in Opti-MEM media for 24 hours, and then the mediawere replaced with Dulbecco's Modified Eagle's Medium (Gibco) containing10% fetal bovine serum (Gibco) and 100 units/ml penicillin 100 μg/mlstreptomycin, and 24 hours after media replacement, SRD5A2 expressionwas examined at the protein level. As the result of repeatedlyconducting two experiments, it was confirmed that, as the treatmentconcentrations of cp-asiSRD5A2 #59(4,4) and #62(4,4) increased, theprotein expression of the target gene SRD5A2 was reduced (see FIG. 9).

[Example 10] Screening for 118 Kinds of RNAi-Inducing Double-StrandedNucleic Acid Molecules Targeting AR

To obtain high-efficiency RNAi-inducing double-stranded nucleic acidmolecules targeting AR, the target sequence of the AR gene was selectedand then asiRNA was designed. The asiRNA structure is different fromthat of generally known siRNAs, and thus when the nucleotide sequencesof asiRNA are designed using a general siRNA design program, it may besomewhat difficult to design an optimized asiRNA. Therefore, asiRNA wasconstructed by the following method. An NCBI db search was used toobtain information on the AR gene (mRNA Accession Number:NM_001011645.2), which is the target gene pertaining to male patternhair loss (androgenetic hair loss). For subsequent animal experiments,nucleotide sequences with at least 80% homology to that of mice weresecured, and then 100 asiRNAs were designed according to a design methodsuch as the exclusion of sequences having a GC content of 30-62% and 4or more G or C consecutive bases, and then synthesized by OliX Inc.(Korea). The synthesized sense and antisense strand RNA oligonucleotideswere annealed at 95° C. for 2 minutes through incubation at 37° C. for 1hour, and the asiRNA annealed by 10% polyacrylamide gel electrophoresis(PAGE) was confirmed using a UV transilluminator.

TABLE 6 118 strands of asiRNA nucleotide sequencestargeting androgen receptor Sequence (5′-3′) No. Name S (16mer)As (21mer)   1 asiAR1 GAGAUGAAGCUUCUGG CCAGAAGCUUCAUCUCCACAG(SEQ ID NO: 429) (SEQ ID NO: 547)   2 asiAR2 GGAGAUGAAGCUUCUGCAGAAGCUUCAUCUCCACAGA (SEQ ID NO: 430) (SEQ ID NO: 548)   3 asiAR3GUGGAGAUGAAGCUUC GAAGCUUCAUCUCCACAGAUC (SEQ ID NO: 431) (SEQ ID NO: 549)  4 asiAR4 UGUGGAGAUGAAGCUU AAGCUUCAUCUCCACAGAUCA (SEQ ID NO: 432)(SEQ ID NO: 550)   5 asiAR5 UCUGUGGAGAUGAAGC GCUUCAUCUCCACAGAUCAGG(SEQ ID NO: 433) (SEQ ID NO: 551)   6 asiAR6 UGAUCUGUGGAGAUGAUCAUCUCCACAGAUCAGGCAG (SEQ ID NO: 434) (SEQ ID NO: 552)   7 asiAR7CUGAUCUGUGGAGAUG CAUCUCCACAGAUCAGGCAGG (SEQ ID NO: 435) (SEQ ID NO: 553)  8 asiAR8 AAGACCUGCCUGAUCU AGAUCAGGCAGGUCUUCUGGG (SEQ ID NO: 436)(SEQ ID NO: 554)   9 asiAR9 UUUCCACCCCAGAAGA UCUUCUGGGGUGGAAAGUAAU(SEQ ID NO: 437) (SEQ ID NO: 555)  10 asiAR10 ACUUUCCACCCCAGAAUUCUGGGGUGGAAAGUAAUAG (SEQ ID NO: 438) (SEQ ID NO: 556)  11 asiAR11AAGGGAAACAGAAGUA UACUUCUGUUUCCCUUCAGCG (SEQ ID NO: 439) (SEQ ID NO: 557) 12 asiAR12 GAAGGGAAACAGAAGU ACUUCUGUUUCCCUUCAGCGG (SEQ ID NO: 440)(SEQ ID NO: 558)  13 asiAR13 CUGAAGGGAAACAGAA UUCUGUUUCCCUUCAGCGGCU(SEQ ID NO: 441) (SEQ ID NO: 559)  14 asiAR14 CAAAAGAGCCGCUGAAUUCAGCGGCUCUUUUGAAGAA (SEQ ID NO: 442) (SEQ ID NO: 560)  15 asiAR15UCAAAAGAGCCGCUGA UCAGCGGCUCUUUUGAAGAAG (SEQ ID NO: 443) (SEQ ID NO: 561) 16 asiAR16 CUUCAAAAGAGCCGCU AGCGGCUCUUUUGAAGAAGAC (SEQ ID NO: 444)(SEQ ID NO: 562)  17 asiSR17 CUUCUUCAAAAGAGCC GGCUCUUUUGAAGAAGACCUU(SEQ ID NO: 445) (SEQ ID NO: 563)  18 asiAR18 UCUUCUUCAAAAGAGCGCUCUUUUGAAGAAGACCUUG (SEQ ID NO: 446) (SEQ ID NO: 564)  19 asiAR19AGGUCUUCUUCAAAAG CUUUUGAAGAAGACCUUGCAG (SEQ ID NO: 447) (SEQ ID NO: 565) 20 asiAR20 AACCAGGGAUGACUCU AGAGUCAUCCCUGCUUCAUAA (SEQ ID NO: 448)(SEQ ID NO: 566)  21 asiAR21 UGAAGCAGGGAUGACU AGUCAUCCCUGCUUCAUAACA(SEQ ID NO: 449) (SEQ ID NO: 567)  22 asiAR22 UUAUGAAGCAGGGAUGCAUCCCUGCUUCAUAACAUUU (SEQ ID NO: 450) (SEQ ID NO: 568)  23 asiAR23UGUUAUGAAGCAGGGA UCCCUGCUUCAUAACAUUUCC (SEQ ID NO: 451) (SEQ ID NO: 569) 24 asiAR24 AUGUUAUGAAGCAGGG CCCUGCUUCAUAACAUUUCCG (SEQ ID NO: 452)(SEQ ID NO: 570)  25 asiAR25 GCUAUGAAUGUCAGCC GGCUGACAUUCAUAGCCUUCA(SEQ ID NO: 453) (SEQ ID NO: 571)  26 asiAR26 GGCUAUGAAUGUCAGCGCUGACAUUCAUAGCCUUCAA (SEQ ID NO: 454) (SEQ ID NO: 572)  27 asiAR27GAAGGCUAUGAAUGUC GACAUUCAUAGCCUUCAAUGU (SEQ ID NO: 455) (SEQ ID NO: 573) 28 asiAR28 UUGAAGGCUAUGAAUG CAUUCAUAGCCUUCAAUGUGU (SEQ ID NO: 456)(SEQ ID NO: 574)  29 asiAR29 GAAGCCAUUGAGCCAG CUGGCUCAAUGGCUUCCAGGA(SEQ ID NO: 457) (SEQ ID NO: 575)  30 asiAR30 CUGGCUUCCGCAACUUAAGUUGCGGAAGCCAGGCAAG (SEQ ID NO: 458) (SEQ ID NO: 576)  31 asiAR31UGCCUGGCUUCCGCAA UUGCGGAAGCCAGGCAAGGCC (SEQ ID NO: 459) (SEQ ID NO: 577) 32 asiAR32 AGUGGGCCAAGGCCUU AAGGCCUUGGCCCACUUGACC (SEQ ID NO: 460)(SEQ ID NO: 578)  33 asiAR33 CCAGGAUGCUCUACUU AAGUAGAGCAUCCUGGAGUUG(SEQ ID NO: 481) (5E0 ID NO: 579)  34 asiAR34 UCCAGGAUGCUCUACUAGUAGAGCAUCCUGGAGUUGA (SEQ ID NO: 462) (SEQ ID NO: 580)  35 asiAR35AACUCCAGGAUGCUCU AGAGCAUCCUGGAGUUGACAU (SEQ ID NO: 463) (SEQ ID NO: 581) 36 asiAR36 UACCGCAUGCACAAGU ACUUGUGCAUGCGGUACUCAU (SEQ ID NO: 464)(SEQ ID NO: 582)  37 asiAR37 AGUACCGCAUGCACAA UUGUGCAUGCGGUACUCAUUG(SEQ ID NO: 465) (SEQ ID NO: 583)  38 asiAR38 CAAUGAGUACCGCAUGCAUGCGGUACUCAUUGAAAAC (SEQ ID NO: 466) (SEQ ID NO: 594)  39 asiAR39UCAAUGAGUACCGCAU AUGCGGUACUCAUUGAAAACC (SEQ ID NO: 467) (SEQ ID NO: 585) 40 asiAR40 UUCAAUGAGUACCGCA UGCGGUACUCAUUGAAAACCA (SEQ ID NO: 468)(SEQ ID NO: 586)  41 asiAR41 UUGGAUGGCUCCAAAU AUUUGGAGCCAUCCAAACUCU(SEQ ID NO: 469) (SEQ ID NO: 587)  42 asiAR42 AGUUUGGAUGGCUCCAUGGAGCCAUCCAAACUCCUGA (SEQ ID NO: 470) (SEQ ID NO: 588)  43 asiAR43AGAGUUUGGAUGGCUC GAGCCAUCCAAACUCUUGAGA (SEQ ID NO: 471) (SEQ ID NO: 589) 44 asiAR44 UCAAGGAACUCGAUCG CGAUCGAGUUCCUUGAUGUAG (SEQ ID NO: 472)(SEQ ID NO: 590)  45 asiAR45 CAUCAAGGAACUCGAU AUCGAGUUCCUUGAUGUAGUU(SEQ ID NO: 473) (SEQ ID NO: 591)  46 asiAR46 CUACAUCAAGGAACUCGAGUUCCUUGAUGUAGUUCAU (SEQ ID NO: 474) (SEQ ID NO: 592)  47 asiAR47GAACUACAUCAAGGAA UUCCUUGAUGUAGUUCAUUCG (SEQ ID NO: 475) (SEQ ID NO: 593) 48 asiAR48 CUUCGAAUGAACUACA UGUAGUUCAUUCGAAGUUCAU (SEQ ID NO: 476)(SEQ ID NO: 594)  49 asiAR49 UGAACUUCGAAUGAAC GUUCAUUCGAAGUUCAUCAAA(SEQ ID NO: 477) (SEQ ID NO: 595)  50 asiAR50 UGAUGAACUUCGAAUGCAUUCGAAGUUCAUCAAAGAA (SEQ ID NO: 478) (SEQ ID NO: 596)  51 asiAR51GGGCUGAAAAAUCAAA UUUGAUUUUUCAGCCCAUCCA (SEQ ID NO: 479) (SEQ ID NO: 597) 52 asiAR52 GAUGGGCUGAAAAAUC GAUUUUUCAGCCCAUCCACUG (SEQ ID NO: 480)(SEQ ID NO: 598)  53 asiAR53 UAUUCCAGUGGAUGGG CCCAUCCACUGGAAUAAUGCU(SEQ ID NO: 481) (SEQ ID NO: 599)  54 asiAR54 CAUUAUUCCAGUGGAUAUCCACUGGAAUAAUGCUGAA (SEQ ID NO: 482) (SEQ ID NO: 600)  55 asiAR55AGCAUUAUUCCAGUGG CCACUGGAAUAAUGCUGAAGA (SEQ ID NO: 483) (SEQ ID NO: 601) 56 asiAR56 UUCAGCAUUAUUCCAG CUGGAAUAAUGCUGAAGAGAG (SEQ ID NO: 484)(SEQ ID NO: 602)  57 asiAR57 CUCUUCAGCAUUAUUC GAAUAAUGCUGAAGAGAGCAG(SEQ ID NO: 485) (SEQ ID NO: 603)  58 asiAR58 CUGCUCUCUUCAGCAUAUGCUGAAGAGAGCAGUGCUU (SEQ ID NO: 486) (SEQ ID NO: 604)  59 asiAR59AAGCACUGCUCUCUUC GAAGAGAGCAGUGCUUUCAUG (SEQ ID NO: 487) (SEQ ID NO: 605) 60 asiAR60 GAAAGCACUGCUCUCU AGAGAGCAGUGCUUUCAUGCA (SEQ ID NO: 488)(SEQ ID NO: 606)  61 asiAR61 CAUGAAAGCACUGCUC GAGCAGUGCUUUCAUGCACAG(SEQ ID NO: 489) (SEQ ID NO: 607)  62 asiAR62 GUGCAUGAAAGCACUGCAGUGCUUUCAUGCACAGGAA (SEQ ID NO: 490) (SEQ ID NO: 608)  63 asiAR63UUCCUGUGCAUGAAAG CUUUCAUGCACAGGAAUUCCU (SEQ ID NO: 491) (SEQ ID NO: 609) 64 asiAR64 GAAUUCCUGUGCAUGA UCAUGCACAGGAAUUCCUGGG (SEQ ID NO: 492)(SEQ ID NO: 610)  65 asiAR65 AGGAAUUCCUGUGCAU AUGCACAGGAAUUCCUGGGGG(SEQ ID NO: 493) (SEQ ID NO: 611)  66 asiAR66 UCACCAAGCUCCUGGAUCCAGGAGCUUGGUGAGCUGG (SEQ ID NO: 494) (SEQ ID NO: 612)  67 asiAR67ACCAGCUCACCAAGCU AGCUUGGUGAGCUGGUAGAAG (SEQ ID NO: 495) (SEQ ID NO: 613) 68 asiAR68 CUACCAGCUCACCAAG CUUGGUGAGCUGGUAGAAGCG (SEQ ID NO: 496)(SEQ ID NO: 614)  69 asiAR69 ACCUGCUAAUCAAGUC GACUUGAUUAGCAGGUCAAAA(SEQ ID NO: 497) (SEQ ID NO: 615)  70 asiAR70 GACCUGCUAAUCAAGUACUUGAUUAGCAGGUCAAAAG (SEQ ID NO: 498) (SEQ ID NO: 616)  71 asiAR71UUUGACCUGCUAAUCA UGAUUAGCAGGUCAAAAGUGA (SEQ ID NO: 499) (SEQ ID NO: 617) 72 asiAR72 CUUUUGACCUGCUAAU AUUAGCAGGUCAAAAGUGAAC (SEQ ID NO: 500)(SEQ ID NO: 618)  73 asiAR73 UCACUUUUGACCUGCU AGCAGGUCAAAAGUGAACUGA(SEQ ID NO: 501) (SEQ ID NO: 619)  74 asiAR74 UUCACUUUUGACCUGCGCAGGUCAAAAGUGAACUGAU (SEQ ID NO: 502) (SEQ ID NO: 620)  75 asiAR75CAGUUCACUUUUGACC GGUCAAAAGUGAACUGAUGCA (SEQ ID NO: 503) (SEQ ID NO: 621) 76 asiAR76 CAUCAGUUCACUUUUG CAAAAGUGAACUGAUGCAGCU (SEQ ID NO: 504)(SEQ ID NO: 622)  77 asiAR77 CUGCAUCAGUUCACUU AAGUGAACUGAUGCAGCUCUC(SEQ ID NO: 505) (SEQ ID NO: 623)  78 asiAR78 GCUGCAUCAGUUCACUAGUGAACUGAUGCAGCUCUCU (SEQ ID NO: 506) (SEQ ID NO: 624)  79 asiAR79CCAUCUAUUUCCACAC GUGUGGAAAUAGAUGGGCUUG (SEQ ID NO: 507) (SEQ ID NO: 625) 80 asiAR80 CCCAUCUAUUUCCACA UGUGGAAAUAGAUGGGCUUGA (SEQ ID NO: 508)(SEQ ID NO: 626)  81 asiAR81 AGCCCAUCUAUUUCCA UGGAAAUAGAUGGGCUUGACU(SEQ ID NO: 509) (SEQ ID NO: 627)  82 asiAR82 UCAAGCCCAUCUAUUUAAAUAGAUGGGCUUGACUUUC (SEQ ID NO: 510) (SEQ ID NO: 628)  83 asiAR83GGAAAGUCAAGCCCAU AUGGGCUUGACUUUCCCAGAA (SEQ ID NO: 511) (SEQ ID NO: 629) 84 asiAR84 CUGGGAAAGUCAAGCC GGCUUGACUUUCCCAGAAAGG (SEQ ID NO: 512)(SEQ ID NO: 630)  85 asiAR85 UUUCUGGGAAAGUCAA UUGACUUUCCCAGAAAGGAUC(SEQ ID NO: 513) (SEQ ID NO: 631)  86 asiAR86 UCCUUUCUGGGAAAGUACUUUCCCAGAAAGGAUCUUG (SEQ ID NO: 514) (SEQ ID NO: 632)  87 asiAR87CCAAGAUCCUUUCUGG CCAGAAAGGAUCUUGGGCACU (SEQ ID NO: 515) (SEQ ID NO: 633) 88 asiAR88 UGCCCAAGAUCCUUUC GAAAGGAUCUUGGGCACUUGC (SEQ ID NO: 516)(SEQ ID NO: 634)  89 asiAR89 AAGUGCCCAAGAUCCU AGGAUCUUGGGCACUUGCACA(SEQ ID NO: 517) (SEQ ID NO: 635)  90 asiAR90 UGCAAGUGCCCAAGAUAUCUUGGGCACUUGCACAGAG (SEQ ID NO: 518) (SEQ ID NO: 636)  91 asiAR91UCUCUGUGCAAGUGCC GGCACUUGCACAGAGAUGAUC (SEQ ID NO: 519) (SEQ ID NO: 637) 92 asiAR92 UCAUCUCUGUGCAAGU ACUUGCACAGAGAUGAUCUCU (SEQ ID NO: 520)(SEQ ID NO: 636)  93 asiAR93 AGAUCAUCUCUGUGCA UGCACAGAGAUGAUCUCUGCC(SEQ ID NO: 521) (SEQ ID NO: 639)  94 asiAR94 CAGAGAUCAUCUCUGUACAGAGAUGAUCUCUGCCAUC (SEQ ID NO: 522) (SEQ ID NO: 640)  95 asiAR95CACUGGCACUAAAAAA UUUUUUAGUGCCAGUGAACAU (SEQ ID NO: 523) (SEQ ID NO: 641) 96 asiAR96 UCACUGGCACUAAAAA UUUUUAGUGCCAGUGAACAUA (SEQ ID NO: 524)(SEQ ID NO: 642)  97 asiAR97 GUUCACUGGCACUAAA UUUAGUGCCAGUGAACAUACA(SEQ ID NO: 525) (SEQ ID NO: 643)  98 asiAR98 UAUGUUCACUGGCACUAGUGCCAGUGAACAUACAUAA (SEQ ID NO: 526) (SEQ ID NO: 644)  99 asiAR99UGUAUGUUCACUGGCA UGCCAGUGAACAUACAUAAAA (SEQ ID NO: 527) (SEQ ID NO: 645)100 asiAR100 UAUGUAUGUUCACUGG CCAGUGAACAUACAUAAAAAU (SEQ ID NO: 528)(SEQ ID NO: 646) 101 asiAR101 GGGUAGUUGCUGAGGU ACCUCAGCAACUACCCAAAGG(SEQ ID NO: 529) (SEQ ID NO: 647) 102 asiAR102 UGGGUAGUUGCUGAGGCCUCAGCAACUACCCAAAGGA (SEQ ID NO: 530) (SEQ ID NO: 648) 103 asiAR103CUUUGGGUAGUUGCUG CAGCAACUACCCAAAGGACAG (SEQ ID NO: 531) (SEQ ID NO: 649)104 asiAR104 CCUUUGGGUAGUUGCU AGCAACUACCCAAAGGACAGA (SEQ ID NO: 532)(SEQ ID NO: 650) 105 asiAR105 CCACCAUCCACAUGAU AUCAUGUGGAUGGUGGACAUA(SEQ ID NO: 533) (SEQ ID NO: 651) 106 asiAR106 CAUUAGUGCCUCUUUGCAAAGAGGCACUAAUGCUUGC (SEQ ID NO: 534) (SEQ ID NO: 652) 107 asiAR107GCAUUAGUGCCUCUUU AAAGAGGCACUAAUGCUUGCU (SEQ ID NO: 535) (SEQ ID NO: 653)108 asiAR108 AGCAUUAGUGCCUCUU AAGAGGCACUAAUGCUUGCUC (SEQ ID NO: 536)(SEQ ID NO: 654) 109 asiAR109 AAGCAUUAGUGCCUCU AGAGGCACUAAUGCUUGCUCC(SEQ ID NO: 537) (SRI ID NO: 655) 110 asiAR110 GCCCAUGUUAGCUUAUAUAAGCUAACAUGGGCACUAG (SEQ ID NO: 538) (SEQ ID NO: 656) 111 asiAR111GAAACUUGUUUGUUGG CCAACAAACAAGUUUCUGCCA (SEQ ID NO: 539) (SEQ ID NO: 657)112 asiAR112 GCAGAAACUUGUUUGU ACAAACAAGUUUCUGCCAUUU (SEQ ID NO: 540)(SEQ ID NO: 658) 113 asiAR113 AUGGCAGAAACUUGUU AACAAGUUUCUGCCAUUUUUA(SEQ ID NO: 541) (SEQ ID NO: 659) 114 asiAR114 AAUGGCAGAAACUUGUACAAGUUUCUGCCAUUUUUAA (SEQ ID NO: 542) (SEQ ID NO: 660) 115 asiAR115GGAAUCUUUUGUUGCU AGCAACAAAAGAUUCCAAGAU (SEQ ID NO: 543) (SEQ ID NO: 661)116 asiAR116 UGGAAUCUUUUGUUGC GCAACAAAAGAUUCCAAGAUU (SEQ ID NO: 544)(SEQ ID NO: 662) 117 asiAR117 UAGUGUUCUGUUCUCU AGAGAACAGAACACUAGCGCU(SEQ ID NO: 545) (SEQ ID NO: 663) 118 asiAR118 CUAGUGUUCUGUUCUCGAGAACAGAACACUAGCGCUU (SEQ ID NO: 546) (SEQ ID NO: 664)

[Example 11] Screening for RNAi-Inducing Double-Stranded Nucleic AcidMolecules Targeting AR

To confirm gene inhibitory efficiency at the mRNA level, the 118selected asiRNAs were transfected into an A549 cell line at aconcentration of 0.3 nM, and qRT-PCR was performed to measure theexpression level of AR mRNA.

The A549 cell line was cultured in Dulbecco's Modified Eagle's Medium(DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100units/ml of penicillin 100 μg/ml of streptomycin. A549 cells were seededin a 96-well plate at a density of 5×10³ cells/well, and a transfectionexperiment was conducted using asiRNA (0.3 nM, OliX PharmaceuticalsInc.) and RNAiMAX (1 μl/ml, Invitrogen Inc.) in Opti-MEM (a total volumeof 100 μl) in accordance with Invitrogen's protocol. After 24 hours, RNApurification and cDNA synthesis were performed in accordance with abasic protocol provided by TOYOBO SuperPrep, the expression level of theAR gene was examined with an AR TaqMan probe (T) using a Bio-RadCFX-4000 machine. First, 88 kinds of asiRNA from among the 118 kinds ofasiRNA were subjected to an asiRNA screening experiment and the 13top-ranked asiRNAs (in Table 6, No. 43, 49, 67, 70, 72, 74, 75, 77, 78,79, 81, 82, and 87) were selected on the basis of inhibitory efficacyagainst the expression of the target gene, and the 13 selected asiRNAsand the 30 remaining asiRNAs (in Table 6, Nos. 88 to 118) were subjectedto a secondary asiRNA screening experiment (see FIGS. 10A and 10B).

The 20 top-ranked asiRNAs (in Table 6, No. 43, 49, 70, 72, 74, 75, 77,78, 79, 81, 82, 87, 89, 90, 93, 96, 106, 110, 111, and 118) having gonethrough secondary asiRNA screening were selected on the basis ofinhibitory efficacy against the expression of the target gene, and anexperiment for confirming the inhibitory effect of the 20 selectedasiRNAs against AR expression at the protein level was performed. A549cells were seeded in a 12-well plate at a density of 5×10⁴ cells/well,and then a transfection experiment was conducted using asiRNA (0.3 nM,OliX Pharmaceuticals Inc.) and RNAiMAX (1 μl/ml, Invitrogen Inc.) inOpti-MEM (a total volume of 1 ml) in accordance with Invitrogen'sprotocol. After 48 hours, the cells were lysed using a mammalian proteinextraction buffer (GE healthcare), and then proteins were quantifiedusing a Bradford assay. 20 μg of the protein of each sample waselectrophoresed using 10% SDS-PAGE at 80 V for 20 minutes and at 120 Vfor 1 hour, and then transferred onto a PVDE membrane (Bio-Rad) at 300mA for 1 hour. After transfer, the membrane was blocked in 5% skim milkfor 1 hour and allowed to react with AR antibody (ABcam, ab133273) at aratio of 1:2000 for 12 hours. The next day, the resulting membrane wasallowed to react with anti-Rabbit HRP (Santa Cruz) at a ratio of 1:5000for 1 hour, and then the expression levels of the AR protein werecompared with each other using ChemiDoc (Bio-Rad). From the results, the9 top-ranked asiRNAs (No. 70, 72, 78, 81, 82, 90, 110, 111, and 118)capable of more effectively inhibiting AR protein expression wereselected (see FIG. 11).

The 9 top-ranked asiRNA candidates (in Table 6, No. 70, 72, 78, 81, 82,90, 110, 111, and 118) having gone through asiRNA screening wereselected on the basis of inhibitory efficacy against the expression ofthe target gene, and an experiment for confirming the inhibitory effectsof the 9 selected asiRNA candidates against AR expression at the mRNAand protein levels and a lower concentration (0.1 nM) was conducted.A549 cells were seeded in a 12-well plate at a density of 5×10⁴cells/well, and a transfection experiment was conducted using asiRNA andRNAiMAX (1 μl/ml, Invitrogen Inc.) in Opti-MEM (a total volume of 0.5ml) in accordance with Invitrogen's protocol. After 48 hours, total RNAwas extracted using TRIzol (TaKaPa), and then cDNA was synthesized usinga high-capacity cDNA reverse transcription kit (Applied Biosystems), andthe expression level of the AR gene was examined using power SYBR greenPCR master Mix (Applied Biosystems), the primers shown in Table 7 below,and a StepOne real-time PCR system.

TABLE 7 Primer nucleotide sequences Name Sequence (5′-3′) size HumanForward GAG TCA ACG GAT TTG GTC 186 GAPDH GT (SEQ ID NO: 665) ReverseGAC AAG CTT CCC GTT CTC AG (SEQ ID NO: 666) Human ForwardGGG GCT AGA CTG CTC AAC 191 AR TG (SEQ ID NO: 667) ReverseGCC AAG TTT TGG CTG AAG AG (SEQ ID NO: 668)

In addition, A549 cells were seeded in a 12-well plate at a density of5×10⁴ cells/well, and a transfection experiment was conducted usingasiRNA and RNAiMAX (1 μl/ml, Invitrogen Inc.) in Opti-MEM (a totalvolume of 0.5 ml) in accordance with Invitrogen's protocol. After 48hours, the cells were lysed using a mammalian protein extraction buffer(GE healthcare), and then proteins were quantified using a Bradfordassay. 20 μg of the protein of each sample was electrophoresed using 10%SDS-PAGE at 80 V for 20 minutes and at 120 V for 1 hour, and thentransferred onto a PVDE membrane (Bio-Rad) at 300 mA for 1 hour. Aftertransfer, the membrane was blocked in 5% skim milk for 1 hour andallowed to react with AR antibody (ABcam, ab133273) at a ratio of 1:2000for 12 hours. The next day, the resulting membrane was allowed to reactwith anti-Rabbit HRP (Santa Cruz) at a ratio of 1:5000 for 1 hour, andthen the expression levels of the AR protein were compared with eachother using ChemiDoc (Bio-Rad). As the result of the experiment for the9 selected asiRNAs, it was confirmed that asiRNA #72, 78, and 110exhibited gene inhibitory efficiency of 50% or higher efficiently evenat a concentration of 0.1 nM (see FIG. 12).

[Example 12] 9 Kinds of Cp-asiRNA Targeting AR Gene and Having SelfCell-Penetrating Ability

AR cp-asiRNAs (a total of 9 kinds) were designed by applying threemodification patterns to 3 kinds of asiRNA targeting AR according to thenumber and position of 2′OMe (methyl), phosphorothioate bonds (PS), andcholesterol, and then synthesized by Dharmacon. cp-asiRNA enhancesendocytosis efficiency and stability, and thus may penetrate through thecell membrane with high efficiency without the aid of a delivery vehicleto thereby inhibit the expression of the target gene. The synthesizedsense and antisense strand RNA oligonucleotides were annealed at 95° C.for 2 minutes through incubation at 37° C. for 1 hour, and cp-asiRNAsannealed by 10% polyacrylamide gel electrophoresis (PAGE) were confirmedusing a UV transilluminator.

TABLE 8 9 kinds of cp-asiRNA nucleotide sequences targeting AR No. NameSequence (5′-3′)  1 cp-asiAR72 mCUmUUmUGmACmCUmGCmUAm*A*U*chol S  2cp-asiAR72 AUUAGCAGGUCAAAmAmGmU*mG*mA*mA*mC AS(7, 4)  3 cp-asiAR72AUUAGCAGGUCAAAmAmGmU*mG*A*A*C AS(4, 4)  4 cp-asiAR72AUUAGCAGGUCAAAmAmGU*G*A*A*C AS(2, 4)  5 cp-asiAR78mGCmUGmCAmUCmAGmUUmCAm*C*U*chol S  6 cp-asiAR78AGUGAACUGAUGCAmGmCmU*mC*mU*mC*mU AS(7, 4)  7 cp-asiAR78AGUGAACUGAUGCAmGmCmU*mC*U*C*U AS(4, 4)  8 cp-asiAR78AGUGAACUGAUGCAmGmCU*C*U*C*U AS(2, 4)  9 cp-asiAR110mGCmCCmAUmGUmUAmGCmUUm*A*U*chol S 10 cp-asiAR110AUAAGCUAACAUGGmGmCmA*mC*mU*mA*mG AS(7, 4) 11 cp-asiAR110AUAAGCUAACAUGGmGmemA*mC*U*A*G AS(4, 4) 12 cp-asiAR110AUAAGCUAACAUGGmGmCA*C*U*A*G AS(2, 4) m: 2′-O-Methyl RNA, *:phosphorothioated bond, chol: cholesterol

[Example 13] Screening for Cp-asiRNA Targeting AR Gene and Having SelfCell-Penetrating Ability

The inhibitory effects of the 9 kinds of cp-asiRNA shown in Table 8against AR expression were examined. An A549 cell line was incubatedwith 1 μM or 3 μM of each of the 9 cp-asiRNAs in Opti-MEM media for 24hours, and then the media were replaced with Dulbecco's Modified Eagle'sMedium (Gibco) containing 10% fetal bovine serum (Gibco) and 100units/ml penicillin 100 μg/ml streptomycin, and after 24 hours, ARexpression was examined at the mRNA level. As the result of repeatedlyconducting four experiments, it was confirmed that the 9 kinds of ARcp-asiRNA exhibited gene inhibitory efficiency of 50% at a concentrationof 3 μM (see FIG. 13).

Under the same experimental conditions, the inhibitory effects of the 9kinds of cp-asiRNA against AR expression were examined at the proteinlevel in an A549 cell line. The A549 cell line was incubated with 1 μMor 3 μM of each of the 9 cp-asiRNAs in Opti-MEM media for 24 hours, andthen the media were replaced with Dulbecco's Modified Eagle's Medium(Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/mlpenicillin 100 μg/ml streptomycin, and after 24 hours, AR expression wasexamined at the protein level. Among them, cp-asiRNA #72(7,4), #78(7,4)(4,4) (2,4), and #110(7,4) (4,4) exhibited target gene proteinexpression inhibitory efficiency of 50% or higher at a concentration of1 μM on the basis of the band intensity of a no treatment (NT) sampleand a ½ NT sample (see FIG. 14).

While the present invention has been described in detail with referenceto specific embodiments thereof, it will be obvious to those of ordinaryskill in the art that these embodiments are provided for illustrativepurposes only and are not intended to limit the scope of the presentinvention. Therefore, the actual scope of the present invention will bedefined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

A 5α-reductase type 1-encoding gene, a 5α-reductase type 2-encodinggene, and an androgen receptor-encoding gene, which play a major role ininhibiting the synthesis of proteins required for hair follicle growthin male pattern hair loss and inducing hair loss by reducing the size ofthe dermal papilla, were selected as target genes, and asymmetric siRNAwith high inhibitory efficiency against each target gene was selected.siRNA according to the present invention exhibits the ability to inhibitthe expression of the target gene for 5α-reductase type 1, 5α-reductasetype 2, or an androgen receptor, and thus may be effectively used as anagent for preventing or treating hair loss.

While the present invention has been particularly shown and describedwith reference to specific embodiments thereof, it will be obvious tothose of ordinary skill in the art that such embodiments are providedfor illustrative purposes only and are not intended to limit the scopeof the present invention. Therefore, the actual scope of the presentinvention should be defined by the appended claims and equivalentsthereof.

SEQUENCE LIST FREE TEXT

Electronic files attached.

1. siRNA specifically binding to mRNA of a 3-oxo-5-alpha-steroid4-dehydrogenase 1 (SRD5A1)-encoding gene having SEQ ID NOs: 669, 670 or671, mRNA of a 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2)-encodinggene having SEQ ID NO: 672, or mRNA of an androgen receptor(AR)-encoding gene having SEQ ID NO: 673 and comprising a sense strandhaving a length of 15 nt to 17 nt and an antisense strand complementaryto the sense strand and having a length of 19 nt or more, wherein a3′-terminus of the sense strand and a 5′-terminus of the antisensestrand form a blunt end.
 2. The siRNA according to claim 1, wherein thesiRNA comprises a sense strand having one selected from SEQ ID NOS: 5,6, 15, 18, 40, 48, 49, 59, 62, 69, 77, 86, 205, 208, 228, 231, 232, 233,237, 238, 239, 240, 242, 248, 249, 259, 260, 262, 265, 283, 284, 285,291, 292, 300, 471, 477, 498, 500, 502, 503, 505, 506, 507, 509, 510,515, 517, 518, 521, 524, 534, 538, 539, and 546 and an antisense strandcomplementary to the sense strand.
 3. The siRNA according to claim 2,wherein the siRNA comprises a sense strand having one selected from thegroup consisting of SEQ ID NOS: 48, 49, 69, 86, 231, 259, 260, 262, 498,500, 506, 509, 510, 518, 538, 539, and 546 and an antisense strandcomplementary to the sense strand.
 4. The siRNA according to claim 1,wherein the antisense strand has a length of 19 nt to 24 nt.
 5. ThesiRNA according to claim 1, wherein the antisense strand is selectedfrom the group consisting of SEQ ID NOS: 105, 106, 115, 118, 140, 148,149, 159, 162, 169, 177, 186, 317, 320, 340, 343, 344, 345, 349, 350,351, 352, 354, 360, 361, 371, 372, 374, 377, 395, 396, 397, 403, 404,412, 589, 595, 616, 618, 620, 621, 623, 624, 625, 627, 628, 633, 635,636, 639, 642, 652, 656, 657, and
 664. 6. The siRNA according to claim5, wherein the antisense strand is selected from the group consisting ofSEQ ID NOS: 148, 149, 169, 186, 343, 371, 372, 374, 616, 618, 624, 627,628, 636, 656, 657, and
 664. 7. The siRNA according to claim 1, whereinthe sense strand or antisense strand of the siRNA comprises at least onechemical modification.
 8. The siRNA according to claim 7, wherein thechemical modification comprises at least one selected from the groupconsisting of: a modification in which an —OH group at a 2′ carbonposition of a sugar structure in a nucleotide is substituted with —CH₃(methyl), —OCH₃ (methoxy), —NH₂, —F (fluorine),—O-2-methoxyethyl-O-propyl, —O-2-methylthioethyl, —O-3-aminopropyl, or—O-3-dimethylaminopropyl; a modification in which oxygen in a sugarstructure in a nucleotide is substituted with sulfur; a modification ofa nucleotide bond to a phosphorothioate, boranophosphate or methylphosphonate; a modification to peptide nucleic acid (PNA), lockednucleic acid (LNA), or unlocked nucleic acid (UNA); and cholesterol orcell-penetrating peptide binding.
 9. The siRNA according to claim 7,wherein the chemical modification is substitution of an —OH group at a2′ carbon position of a sugar structure in a nucleotide with —CH₃(methyl), modification of a nucleotide bond to a phosphorothioate, orcholesterol binding.
 10. The siRNA according to claim 9, wherein thechemical modification comprises at least one selected from the groupconsisting of: a modification in which the —OH group at the 2′ carbonposition of a sugar structure in the 5′- or 3′-terminus nucleotide ofthe sense strand is substituted with —CH₃ (methyl); a modification inwhich the —OH group at the 2′ carbon position of a sugar structure intwo or more nucleotides of the sense strand or the antisense strand issubstituted with —CH₃ (methyl); a modification of 25% or more of bondsbetween nucleotides in the sense or antisense strand tophosphorothioate; and cholesterol binding at the 3′-terminus of thesense strand.
 11. A composition for preventing or treating hair loss,the composition comprising the siRNA according to claim 1.